CN109069561A

CN109069561A - Oncolytic virus and checkpoint inhibitor combination treatment

Info

Publication number: CN109069561A
Application number: CN201780006365.7A
Authority: CN
Inventors: 布莱恩·利克蒂; 约翰·贝尔
Original assignee: Turnstone LP
Current assignee: Turnstone LP
Priority date: 2016-01-11
Filing date: 2017-01-11
Publication date: 2018-12-21
Also published as: MX2018008346A; BR112018013995A2; EP3402500A4; US20190022203A1; WO2017120670A1; JP2019501205A; US20190070280A1; CA3011157A1; KR20190038470A; AU2017207532A1; EP3402500A1

Abstract

The present invention relates to the combination for simultaneously, separately or sequentially using and its for the purposes for the treatment of cancer, the combination includes (a) oncolytic virus and the checkpoint (b) inhibitor.

Description

Oncolytic virus and checkpoint inhibitor combination treatment

Technical field

The present invention relates generally to virology and medicine.In some aspects, the present invention relates to (especially molten by oncolytic virus Tumor rhabdovirus) and checkpoint inhibitor be used for treating cancer combination treatment.

Background technique

Oncolytic virus specifically infects, replicates and kill malignant cell wherein, keeps normal tissue unaffected.It is several Oncolytic virus has reached the advanced stage of the clinical assessment for treating various tumours.

The rhabdovirus of display oncolytic activity, including vesicular stomatitis virus (vesicular has been described Stomatitis virus, VSV) and Maraba virus.It can be by increasing the prominent of the viral sensibility to host immune response Become, further enhances the intrinsic tumour taxis (oncotropism) of these viruses.

The effect of oncolytic virus, depends not only on their cell lysis activity, and additionally depending on them stimulates antineoplastic immune Ability.A kind of method for improving the Clinical efficacy of oncolytic virus is that tumour antigen is expressed from virus.Therefore, it has proved that The engineered VSV to express tumour antigen can be used as oncolytic virus immunotherapy.It has been shown that by first in the VSV of transformation Apply tumour antigen before to cause antineoplastic immune, and then application expresses the oncolytic virus of identical tumour antigen to add Strong already present antineoplastic immune enhances the antitumor efficacy (Bridle of the VSV of expression tumour antigen wherebyet al., Mol. Ther., 18(8):1430-1439 (2010))。

Need to improve the further method of oncolytic virus effect.

Summary of the invention

It has been found by the present inventors that relative to any medicament is administered alone, oncolytic virus and immunologic test point inhibitor are total With clinically relevant cancer model is applied to, the surprising increase of the stimulation of antigenspecific T lymphocyte will lead to, along with Significant survival benefit.Therefore, in some embodiments, this application provides for cancer treatment and/or prevention and/or Transfer stove is established in mammals and/or the combination for starting, enhancing or extending antitumor reaction in mammals is treated Method, the combination including (i) oncolytic virus and (ii) one or more immunologic test point inhibitor are co-administered to mammal.? In some terms, oncolytic virus and immunologic test point inhibitor is co-administered to the object with cancer compared with individual treatment, Provide antineoplastic immune that is enhancing and even cooperateing with.In related fields, the antitumor action of combination treatment is even in disease Poison still has after removing, and can extend to one or more tumours being uninfected by.In other related fields, provide A kind of effect for enhancing, reinforcing or extend checkpoint inhibitor or the toxicity or dosage or treatment number of times that make checkpoint inhibitor The method that can be reduced, including (i) oncolytic virus and (ii) one or more immune inspections are applied to mammal in need Make an inventory of the combination of inhibitor.

It in some embodiments, is the oncolytic bullet shape disease with replication capacity according to the oncolytic virus of the combination treatment Poison.Such oncolytic rhabdovirus includes but is not limited to wild type or Arajas virus, Chandipura disease through gene modification Poison, Cocal virus, Isfahan virus, Maraba virus, Piry virus, vesicular stomatitis Alagoas virus, BeAn 157575 viruses, Boteke virus, Calchaqui virus, America eel virus (Eel virus American), Grey Lodge Virus, Jurona virus, Klamath virus, Kwatta virus, La Joya virus, Malpais Spring virus, Mount Elgon bat viruses, Perinet virus, Tupaia virus, Farmington, Bahia Grande virus, Muir Springs Viral, Reed Ranch virus, Hart Park are viral, Flanders virus, Kamese is viral, Mosqueiro is viral, Mossuril virus, Barur virus, Fukuoka (Fukuoka) virus, Kern valley (Kern Canyon) virus, Nkolbisson Viral, Le Dantec virus, Keuraliba are viral, Connecticut virus, New Minto is viral, Sawgrass is viral, Chaco is viral, Sena Madureira virus, Timbo virus, Almpiwar virus, Aruac is viral, Bangoran is viral, Bimbo virus, Bivens Arm are viral, Blue crab virus, Charleville is viral, Coastal Plains is viral, 7292 virus of DakArK, Entamoeba virus, Garba virus, Gossas virus, Humpty Doo virus, Joinjakaka Viral, Kannamangalam virus, Kolongo are viral, Koolpinyah virus, Kotonkon is viral, Landji is viral, Manitoba is viral, Marco virus, Nasoule virus, Navarro virus, Ngaingan is viral, Oak-Vale is viral, Obodhiang virus, Oita are viral, Ouango virus, Parry Creek is viral, Rio Grande cichlid is viral, Sandjimba virus, Sigma are viral, Sripur virus, Sweetwater Branch is viral, Tibrogargan is viral, Xiburema virus, Yata virus, Rhode Island, Adelaide River virus, Berrimah virus, Kimberley Virus or bovine ephemeral fever virus (Bovine ephemeral fever virus).In some preferred embodiments, oncolytic Rhabdovirus is the Vesiculovirus of wild type or recombination.In other preferred embodiments, oncolytic rhabdovirus is wild type Or VSV, Farmington, Maraba, Carajas, Muir Springs or the Bahia grande virus of recombination, including its change Allosome.In particularly preferred embodiments, oncolytic rhabdovirus is VSV or Maraba rhabdovirus.It is particularly preferred at other Embodiment in, oncolytic rhabdovirus is VSV or Maraba rhabdovirus, it includes it is one or more increase virus tumours The gene modification of selectivity and/or oncolysis.

In relevant embodiment, it is modified according to the oncolytic virus of combination treatment anti-to express one or more tumours Original, such as [0071]-[0082] section and U.S. Patent Application Publication No. 2012/ in WIPO publication number WO 2014/127478 Those of mentioned in 0014990 [0042] section, content is incorporated herein by reference.In preferred embodiments, molten Tumor virus is oncolytic rhabdovirus (such as VSV or Maraba bacterial strain), expression MAGEA3, human papilloma virus E6/E7 fusion Albumen, six cross-film epithelium antigens of human prostate albumen or Cancer Testis Antigens 1 or its variant.In particularly preferred implementation In scheme, oncolytic virus is the oncolytic rhabdovirus selected from Maraba MG1 and VSVdelta51, expresses MAGEA3, human milk head Tumor virus E6/E7 fusion protein, six cross-film epithelium antigens of human prostate albumen or Cancer Testis Antigens 1 or its variant.

In some respects, it provides for treating in mammals and/or the combination treatment of pre- anti-cancer comprising to Mammal be co-administered (i) express tumour antigen oncolytic rhabdovirus (such as VSVdelta51 or Maraba MG1) and (ii) checkpoint inhibitor (such as monoclonal antibody of anti-CTLA 4 or PD-1/PD-L1), the mammal is to the tumour antigen With pre-existing immunity, the tumour antigen is selected from MAGEA3, human papilloma virus E6/E7 fusion protein, people forefront The six cross-film epithelium antigens or cancer testis antigen 1 of gland albumen or its variant.In preferred embodiments, in mammal The pre-existing immunity is by carrying out immunity inoculation to mammal with tumour antigen before applying oncolytic virus Come what is established.In the relevant embodiments, before the first time dosage of the oncolytic rhabdovirus of expression tumour antigen, application inspection Make an inventory of the first time dosage of inhibitor, and can the oncolytic rhabdovirus of expression tumour antigen first time (or second, Third time etc.) application after, application checkpoint inhibitor with post dose.

In combined other side described herein, oncolytic rhabdovirus expresses checkpoint inhibitor (for example, oncolytic bullet Shape expressing viral inhibits the single-chain antibody of albumen for checkpoint), and it is related optionally also to express tumour as described herein Antigen.

The combined oncolytic virus can be used as 10,100,10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、 10¹²、10¹³、10¹⁴Or more one or more dosage application in virion (vp) or plaque forming unit (pfu).Excellent In the embodiment of choosing, oncolytic virus is oncolytic rhabdovirus, e.g. optionally expresses the open country of one or more tumour antigens The VSV or Maraba of raw type or gene modification, and with 10⁶-10¹⁴pfu、10⁶-10¹²pfu、10⁸-10¹⁴pfu、10⁸-10¹²Or 10¹⁰-10¹²One or more dosage in pfu or any range therebetween are applied to the mankind with cancer.Application can lead to It crosses in peritonaeum, intravenous, intra-arterial, intramuscular, intradermal, subcutaneous or intranasal administration.In preferred embodiments, oncolytic virus By systemic administration, especially by intravascular application comprising injection, perfusion etc..

In some respects, the combined checkpoint inhibitor is biopharmaceuticals or small molecule.On the other hand, it checks Point inhibitor is monoclonal antibody, humanized antibody, human antibodies, fusion protein or combinations thereof.On the other hand, checkpoint presses down Preparation inhibits checkpoint albumen, and the checkpoint albumen includes but is not limited to cytotoxic lymphocyte antigen -4 (cytotoxic T-lymphocyte antigen-4, CTLA4), apoptosis albumen 1(programmed cell Death protein 1, PD-1) and its ligand PD-L1 and PD-L2, B7-H3, B7-H4, herpesviral enter regulator (herpesvirus entry mediator, HVEM), T cell memebrane protein 3(T cell membrane protein 3, TIM3), galactose agglutinin 9(galectin 9, GAL9), lymphocyte activation gene 3(lymphocyte activation Gene 3, LAG3), containing V- domain immunoglobulin (Ig) T cell activation repressor (V-domain Immunoglobulin (Ig)-containing suppressor of T-cell activation, VISTA), killing it is thin Born of the same parents' immunoglobulin-like receptor (Killer-Cell Immunoglobulin-Like Receptor, KIR), B and T lymphocyte Attenuator (B and T lymphocyte attenuator, BTLA), the T cell immunity receptor with Ig and ITIM structural domain (T cell immunoreceptor with Ig and ITIM domain, TIGIT) or combinations thereof.On the other hand, it checks Point inhibitor and checkpoint albumen ligand interact comprising but be not limited to CTLA4, PD-1, B7-H3, B7-H4, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, BTLA, TIGIT or combinations thereof.

In some preferred embodiments, oncolytic virus (such as oncolytic rhabdovirus) and the checkpoint CTLA4 inhibitor It is co-administered.The checkpoint CTLA4 inhibitor includes but is not limited to monoclonal antibody, such as easy Puli's nurse Ma (Ipilimumab) (Yervoy;BMS) and Tremelimumab(AstraZeneca/MedImmune).

In other preferred embodiments, oncolytic virus (such as oncolytic rhabdovirus) and PD-1 or its ligand (PD- L1 inhibitor) is co-administered.The checkpoint PD-1/PD-L1 inhibitor includes but is not limited to: for the monoclonal antibody of PD-1, Such as receive military monoclonal antibody (Nivolumab) (Opdivo; Bristol-Myers Squibb;Code name BMS-936558), pyridine aldoxime methyliodide (PAM) list Anti- (Pembrolizumab) (Keytruda) and Pidilizumab；Anti- PD-1 fusion protein, such as AMP-224(is by PD-L2 Extracellular domain and human IgG1 Fc district's groups at)；And the monoclonal antibody of anti-PD-L1, such as BMS-936559(MDX- 1105), Aunar Zhu monoclonal antibody (Atezolizumab) (Genentech/Roche;MPDL3280A), Durvalumab (AstraZeneca/MedImmune;MEDI4736) and Avelumab(Merck KGaA).

Oncolytic virus (such as oncolytic rhabdovirus) and immunologic test point inhibitor simultaneously or sequentially are applied in need Mammal, and can be used as same preparation a part or with different preparations application.In preferred embodiments, exist Before starting to be treated with checkpoint inhibitor, start to be treated with oncolytic virus.

Cancer according to combined therapy as described herein includes but is not limited to leukaemia, acute lymphoblastic leukemia, urgency Acute myeloid leukemia, myeloblast progranulocyte, Myelomonocyte rubricyte leukocythemia, chronic leukemia, chronic grain are thin Born of the same parents (granulocyte) leukaemia, chronic lymphocytic leukemia, lymphoma mantle cell, primary central nervous system lymphoma, primary It is base spy lymthoma and marginal zone B-cell lymphoma, polycythemia vera lymthoma, Hodgkin's disease, non-Hodgkin lymphoma, multiple Property myeloma, Walden Si Telun (Waldenstrom) macroglobulinemia, heavy chain disease, solid tumor, sarcoma and cancer, fibre Tie up sarcoma, myxosarcoma, embryonal-cell lipoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, leaching Hand shaft sarcoma, lymphatic endothelial cells tumor, synovialoma, celiothelioma, outstanding Yin Shi (Ewing) tumour, leiomyosarcoma, band muscle Tumor, colon sarcoma, colorectal cancer, cancer of pancreas, breast cancer, oophoroma, prostate cancer, squamous cell carcinoma, basal-cell carcinoma, gland Cancer, syringocarcinoma, carcinoma of sebaceous glands, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, cephaloma, lung bronchogenic carcinoma, clear-cell carcinoma, liver Cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Weir Mu Shi (Wilm) tumour, cervical carcinoma, uterine cancer, testis are swollen Tumor, lung cancer, Small Cell Lung Cancer, non-small cell lung cancer, bladder cancer, epithelioma, glioma, astrocytoma, medulloblast Tumor, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neurinoma, oligodendroglioma, hemangioma, mind Through blastoma, retinoblastoma, nasopharyngeal carcinoma, the cancer of the esophagus, basal-cell carcinoma, cancer of bile ducts, bladder cancer, osteocarcinoma, brain and in Pivot nervous system (CNS) cancer, cervical carcinoma, choriocarcinoma, colorectal cancer, connective tissue cancer, Alimentary System, endometrium Cancer, cancer of the esophagus, cancer eye, head and neck cancer, gastric cancer, intraepithelial neoplasia, kidney, laryngocarcinoma, liver cancer, lung cancer (chest cancer) (including Small Cell Lung Cancer, Squamous non-small cell lung cancer and non-squamous non-small cell lung cancer)), melanoma (including metastasis melanin tumor), nerve it is female thin Born of the same parents' tumor；Carcinoma of mouth (such as lip, tongue, mouth and pharynx), oophoroma, cancer of pancreas, retinoblastoma, rhabdomyosarcoma, the carcinoma of the rectum； Respiratory system carcinoma, sarcoma, cutaneum carcinoma, gastric cancer, carcinoma of testis, thyroid cancer, uterine cancer and urinary system cancer.Some preferred In embodiment, cancer to be treated is selected from squamous or non-squamous non-small cell lung cancer (NSCLC), (such as hormone is difficult for breast cancer The property controlled metastatic breast cancer), head and neck cancer (such as head and neck squamous cell carcinoma), metastatic colorectal carcinoma, hormone-sensitive or hormone Refractory prostate cancer, colorectal cancer, oophoroma, hepatocellular carcinoma, clear-cell carcinoma, soft tissue sarcoma and Small Cell Lung Cancer.? In some preferred embodiments, cancer to be treated is ER/PR-, HER2+ breast cancer, three negative (to progesterone receptor, female The expression of hormone receptor and human epidermal growth factor receptor-2 is negative) breast cancer, ER and/or PR+ HER2+ breast cancer, NSCLC(squamous and/or non-squamous) or gastroesophageal junction (GEJ) cancer.

It in one aspect, is the people with cancer with the object of the combined therapy, the cancer is for a kind of or more The treatment of kind of chemotherapeutant is refractory (progress) and/or for being refractory with the treatment of one or more antibody. Checkpoint inhibitor of the invention and oncolytic virus combination can be applied to the people with cancer, and the people is accredited as checkpoint suppression The candidate of preparation for treating.In some embodiments, oncolytic virus is applied to enhance the effect of checkpoint inhibitor for treating, and It is applied before applying checkpoint inhibitor.

In some respects, treatment is determined that the clinical effectiveness is such as, but not limited to: the antitumor work of T cell by clinical effectiveness Property increase, enhancing or extension, antitumor T cell or the quantity of activating T cell increase or its group compared with the quantity before treatment It closes.On the other hand, clinical effectiveness is that tumor stabilisation, tumor regression, tumor regression and/or overall survival increase.

On the other hand, the method further includes: prior to, concurrently with, or after with the combination therapy to treat, to The object application chemotherapeutant, targeted therapy, radiation, cold therapy or thermotherapy therapy.

Related embodiment of the invention provides one kind for treating cancer in mammals or is used to prepare treatment cancer The pharmaceutical composition of the drug of disease, wherein the combination includes oncolytic virus (preferably oncolytic rhabdovirus) and checkpoint inhibitor. In some embodiments, pharmaceutical composition includes and for the people of CTLA4 or PD-1/PD-L1 or Humanized monoclonal antibodies and appoints Selection of land is through modifying the VSV or Maraba plants of rhabdoviruses to increase to the selectivity of cancer cell, such as, but not limited to VSVdelta51 or Maraba MG1.

On the other hand, the kit for inducing immune response in mammals is provided comprising oncolytic virus (preferably oncolytic rhabdovirus) and checkpoint inhibitor.In some embodiments, the kit includes VSV or Maraba bacterium Strain rhabdovirus and checkpoint inhibitor, the rhabdovirus are optionally modified the selectivity to increase to cancer cell, such as But it is not limited to expression MAGEA3, human papilloma virus E6/E7 fusion protein, six cross-film epithelium antigens of human prostate albumen, cancer VSVdelta51 the or Maraba MG1 of disease testis antigen 1 or its variant, the checkpoint inhibitor are preferably PD-1, PD- The checkpoint L1 and/or CTLA-4 inhibitor, and the second virus can be optionally further included, second virus is immune Different from oncolytic rhabdovirus on, allow to as in heterologous prime-boost (prime-boost) immunity inoculation " just exempting from ", and second expressing viral antigen identical with oncolytic rhabdovirus.The kit, which may further include, to be made With the explanation of the treatment of cancer with combinations.

Application discusses other embodiments of the invention.Any embodiment party about one aspect of the present invention discussion Case is also applied for other aspects of the present invention, and vice versa.Embodiment in specific embodiment and embodiment part should manage Solution is the non-limiting embodiments of the invention suitable for all aspects of the invention.

When in claim and/or specification in use, term " inhibition ", " reduction " or " preventing " or these terms Any variation include it is any it is measurable reduction or complete inhibition to realize desired result.Desired result includes but unlimited In alleviation, carcinous or hyperproliferative disorders are reduced, slowed down or eradicated, and are made the life better quality or extension service life.

When being used in combination with the term " includes " in claim and/or specification, word "a" or "an" is used It can indicate "one", but it is also consistent with the meaning of " one or more ", "at least one" and " one or more than one ".

In entire the application, term " about " is for the mark that expression value includes for the error for determining the device or method of value Quasi- deviation.

The use of term "or" only refers to alternative solution unless explicitly stated otherwise or replaces for indicating "and/or" in claim It is excluded each other for scheme, although the disclosure is supported to only relate to the definition of alternative solution and "and/or".

As used in the present description and claims, word "comprising" (and any type of include), " tool Have " (and any type of have), " comprising " (and any type of include) or " containing " (and any type of contain ) it is all inclusiveness or unconfined and be not excluded for other unlisted elements or method and step.

Term " mammal " refers to the mankind and non-human mammal.

" checkpoint inhibitor " used herein refer to act on be TNF receptor or B7 superfamily member surface protein On medicament, the medicament including being bound to negative costimulatory molecules, including but not limited to CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3 and/or their own ligand, including PD-L1.

Term " programmed death 1 ", " apoptosis 1 ", " protein PD-1 ", " PD-1 " and " PD1 " is interchangeable It uses, and variant including people PD-1, isomers, Species homologues and there is at least one common epitope with PD-1 Analog.Complete PD-1 sequence can be found at GenBank accession number U64863.

Term " the T lymphocyte antigen -4 of cytotoxicity ", " CTLA-4 ", " CTLA4 " and " CTLA-4 antigen " is interchangeable Use, and the variant including people CTLA-4, isomers, Species homologues and with CTLA-4 have at least one common table The analog of position.Complete CTLA-4 nucleic acid sequence can be found at GenBank accession number L15006.

It should be understood that while " combination treatment " contemplates the combined component, sequentially or separated application.At this The one aspect of invention, " combination treatment ", which contemplates, is administered simultaneously oncolytic virus and checkpoint inhibitor.Of the invention another Aspect, " combination treatment ", which contemplates, sequentially applies oncolytic virus and checkpoint inhibitor.In another aspect of the invention, " connection Close therapy " contemplate separate administration oncolytic virus and checkpoint inhibitor.When sequentially or separate administration oncolytic virus and checkpoint When inhibitor, oncolytic virus and inspection are applied within the time interval for allowing therapeutic agent to show cooperation (such as collaboration) effect Point inhibitor.In preferred embodiments, oncolytic virus and checkpoint inhibitor are in mutual 1,2,3,6,12,24,48,72 Hour within or 4,5,6 or 7 days within or 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24, 25, it is applied within 26,27,28,29,30 or 31 days.In some embodiments, the first time dosage of checkpoint inhibitor it Before (i.e. before starting to be treated with checkpoint inhibitor), the first time dosage for applying oncolytic virus (starts to use oncolytic Virus is treated), or vice versa, and may include carrying out treatment with oncolytic virus and being controlled with checkpoint inhibitor Treat the stage of overlapping.In other embodiments, can while the first time dosage of checkpoint inhibitor or at about, Apply the first time dosage of oncolytic virus.In other embodiments, in the first time dosage of checkpoint inhibitor (or second It is secondary, third time or subsequent dosage) after, apply the first time dosage of oncolytic virus, and may include with oncolytic virus into Row treatment and the stage that treatment overlapping is carried out with checkpoint inhibitor.

From the following detailed description, other objects of the present invention, feature and advantage will become obvious.However, should Understand, although detailed description and specific embodiment show specific embodiments of the present invention, but only give by way of illustration Out, because for those skilled in the art, being obtained from these detailed descriptions each in the spirit and scope of the present invention Kind change and modification are obvious.

Detailed description of the invention

The following drawings forms a part of this specification, and is included to further illustrate certain aspects of the invention. It, can be more preferable by reference to one or more of these attached drawings and the detailed description of combination specific embodiment given herein Ground understands the present invention.

Fig. 1: inhibitor (aCTLA4 in checkpoint is co-administered to the mouse for carrying subcutaneous CT26 tumour；Anti-CTLA 4 antibody) With oncolytic rhabdovirus (MG1 GFP；The Maraba double-mutant (GFP) of expressing green fluorescent protein) therapeutic scheme.1st group (control group) receives PBS；2nd group (MG1/GFP) only in the 3 intravenous injection MG1 GFP of receiving in the 1st, 3 and 5 day；3rd group (MG1/GFP+CTLA4) in the 1st, 3 and 5 day 3 intravenous injection MG1 GFP of receiving and in the 1st, 4,7,10,13,16,19 With the 8 intraperitoneal injection anti-CTLA 4 antibodies of receiving in 22 days.4th group (CTLA4) at the 1st, 4,7,10,13,16,19 and 22 day only Receive 8 intraperitoneal injection anti-CTLA 4 antibodies.In the 10th day progress immunoassay.

The immune response of CT26 specificity at Fig. 2: the 10 day: total IFN-γ response.For each group, show in vitro It is exposed to AH1(immundominance CT26 epitope (gp70_423-431)) percentage of the CD8+ T cell of secretion of gamma-IFN afterwards.MG1/ The co-administration of GFP and CTLA4 increases the percentage of the CD8 T cell of secretion of gamma-IFN in response AH1.

The immune response of CT26 specificity at Fig. 3: the 10 day: the T cell of the IFN-γ list positive.For each group, show It is exposed to AH1(immundominance CT26 epitope (gp70 in vitro_423-431)) the CD8+ T of secretion of gamma-IFN (rather than TNF α) afterwards The percentage of cell.The co-administration of MG1/GFP and CTLA4 increases the CD8+ T cell of the IFN-γ list positive in response AH1 Percentage.

The immune response of CT26 specificity at Fig. 4: the 10 day: the T cell of the bis- positives of IFN-γ/TFN α.For each group, It shows and is exposed to AH1(immundominance CT26 epitope (gp70 in vitro_423-431)) afterwards the CD8+ T of secretion of gamma-IFN and TNF α it is thin The percentage of born of the same parents.The CD8+ T that the co-administration of MG1/GFP and CTLA4 increases the bis- positives of IFN-γ in response AH1/TFN α is thin The percentage of born of the same parents.

Fig. 5: tumor growth curve.Depict the gross tumor volume of the mouse of each treatment group since the 0th day at any time Variation.

Fig. 6: Kaplan-Meier survivorship curve.Depict the survival hundred of the mouse of each treatment group since the 0th day Divide ratio.

Fig. 7: to the mouse for carrying metastatic lung tumors, the adenovirus (Ad-hDCT) of hDCT tumour antigen is being expressed After just exempting from application, checkpoint inhibitor (anti-PD-1 antibody) is co-administered and expresses the oncolytic bullet shape disease of hDCT tumour antigen The therapeutic scheme of malicious (MG1 hDCT).1st group receives PBS(control group)；2nd group (α PD-1) only the 8th, 10,13,15,17, 20,22,24,27, the 29 and 31 days receiving anti-PD-1 antibody of 11 intraperitoneal injections；3rd group (Ad:MG1 hDCT) connect at the 5th day By single 2 × 10⁸The application of the AdhDCT of pfu, then in the 2 intravenous injection MG1 hDCT of receiving in the 14th day and the 17th day. 4th group (Ad:MG1 hDCT+ α PD-1) received single 2 × 10 at the 5th day⁸The application of the AdhDCT of pfu, then (i) the 14th It and the 2 MG1 hDCT of intravenous injection in the 17th day, and (ii) connect at 8,10,13,15,17,20,22,24,27,29 and 31 days By the anti-PD-1 antibody of 11 intraperitoneal injections.14th, 20,27 day progress immunoassay.

Fig. 8 A-8F: just exempting from time to peak point (the 14th day) immunoassay.Fig. 8 A and 8B are illustrated at the 14th day from every The percentage of the lymphocyte of CD8 and CD4 marker stained positive in the PBMC of a treatment group.Fig. 8 C illustrates secretion of gamma-IFN CD8+ T cell percentage (total).Fig. 8 D-8F illustrates the immundominance for being exposed to SVY(DCT in vitro at the 14th day Epitope (DCT_180-188)) after, only secretion of gamma-IFN (Fig. 8 D), secretion of gamma-IFN and TNF α (Fig. 8 E) from each treatment group and Secretion of gamma-IFN, TNF α and IL-2(Fig. 8 F) CD8+ T cell percentage.

Fig. 9 A-9D: the immunoassay of (the 20th day) when peak value is reinforced.Fig. 9 A-9B was illustrated at the 20th day, from each In the PBMC for the treatment of group in the cent lymphocytes (Fig. 9 A) of CD8 marker stained positive and the blood from each treatment group The number (Fig. 9 B) of CD8+ T cell.Fig. 9 C-9D illustrates to secrete IFN- in each treatment group of response SVY at the 20th day The CD8+ T cell of γ account for sum percentage and every microlitre in secretion of gamma-IFN CD8+ T cell account for sum quantity.

Figure 10 A-F: the phenotypic analysis of the T cell of (the 20th day) SVY- specificity when peak value is reinforced.Figure 10 A-10C is illustrated After being exposed to SVY in vitro, only secretion of gamma-IFN from each treatment group (exclude those also TNF secretion α and/or IL-2) (Figure 10 A), secretion of gamma-IFN and TNF α (Figure 10 B) and secretion of gamma-IFN, TNF α and IL-2(Figure 10 C) CD8+ T cell hundred Divide ratio.Figure 10 D-10F illustrates after being exposed to SVY in vitro, every microlitre of only secretion of gamma-IFN in the blood from each treatment group (Figure 10 D), secretion of gamma-IFN and TNF α (Figure 10 E) and secretion of gamma-IFN, TNF α and IL-2(Figure 10 F) CD8+ T cell number Amount.

Figure 11 A-11D: immunoassay-reinforcement later period (the 27th day).Figure 11 A-11B was compared at 27 days, MG1-hDCT Treatment group (" just exempt from: reinforcing ") and combined therapy group (the anti-PD-1 antibody of MG1-hDCT+；" just exempt from: reinforcing PD1 ") in CD8 label The quantity (Figure 11 B) of CD8+ T cell in the percentage (Figure 11 A) and blood of the lymphocyte of object stained positive.Figure 11 C-11D It compares at 27 days, in these treatment groups of response SVY, the CD8+ T cell of secretion of gamma-IFN accounts for the percentage and blood of sum The CD8+ T cell of every microlitre of secretion of gamma-IFN accounts for the quantity of sum in liquid.

Figure 12 A-12F: in the phenotypic analysis for the SVY specific T-cells for reinforcing later period (the 27th day).Figure 12 A-12C explanation After being exposed to SVY in vitro, the only secretion of gamma-IFN from particular treatment group (excludes those also TNF secretion α and/or IL-2 ) (Figure 12 A), secretion of gamma-IFN and TNF α (Figure 12 B) and secretion of gamma-IFN, TNF α and IL12(Figure 12 C) CD8+ T cell Percentage.Figure 12 D-12F is illustrated after being exposed to SVY in vitro, is only secreted in every microlitre of blood from particular treatment group IFN-γ (Figure 12 D), secretion of gamma-IFN and TNF α (Figure 12 E) and secretion of gamma-IFN, TNF α and IL12(Figure 12 F) CD8+ T it is thin The quantity of born of the same parents.

Figure 13: Kaplan-Meier survivorship curve.Depict the survival hundred of the mouse since each processing group the 0th day Divide ratio.

Figure 14 A-C: for Detailed description of the invention compared with individual prime-boost (" no antibody "), the first of hDCT exempts from the same of application When with single dose application anti-PD-1 Antibody on Mouse weight influence (" the 7th day antibody (simultaneously) ") (Figure 14 A), hDCT just Exempt from influence (" the 10th day antibody (sequentially) ") (figure of 3 days anti-PD-1 Antibody on Mouse weight with single dose application after applying 14B), the first influence for exempting to start for 3 days after applying the anti-PD-1 Antibody on Mouse weight applied with multi-dose of hDCT is (" continuous anti- Body (the 10th day starts) ") (Figure 14 C).

Figure 15: for Detailed description of the invention compared with individual prime-boost (" no antibody "), first with hDCT exempts from application on the same day Influence of the anti-PD-1 Antybody therapy (" the 7th day antibody (simultaneously) ") started to Maraba virus titer.

A-16B: Figure 16 A of Figure 16: with MG1-GFP or the MG1-GFP through irradiating is infected 4T1 cell 24 hours with 3 MOI Microarray analysis.Thermal map includes most significant gene of the enrichment more than 4 times compared with non-infected cells.Figure 16 B: MG1-GFP is used Or the MG1-GFP through irradiating infects 24 hours microarray analysis of EMT6 cell with 3 MOI.Thermal map includes and non-infected cells Most significant gene compared to enrichment more than 4 times.

A-17B: Figure 17 A of Figure 17: incubation 24 is small in the culture medium handled through 4T1 through MG1 infection for removing virus When after 4T1 cell surface PDL1 expression flow cytometry.Figure 17 B: with continuous processing 4T1 lotus in MG1-GFP tumour Tumor mouse 5 days.The figure illustrates be used as to adjust in 12 days spleens (left figure) after last time virus injection and tumour (right figure) The percentage of the T cell of property T cell.Double tail non-matching T are examined: * *: p < 0.01.

Continuous processing 5 days in Figure 18 A-18B: Figure 18 A:4T1 tumor-bearing mice MG1-GFP tumour, then every other day abdomen Injection anti-CTLA 4 and each 100 μ g of anti-PD1(in film) combination, 5 injections in total.It collects and measures tumour.For each reality (including 4 experiments in figure) is tested, by each gross tumor volume divided by the mean tumour volume of control animals.Statistical analysis uses The double tail t of non-matching are examined: *: p < 0.01 * * *: p < 0.001 of *: p < 0.05, *.Figure 18 B: model is excited again using tumour The tumour growth (left figure) and Kaplan-Meier survival analysis (right figure) of 4T1 tumor-bearing mice, wherein the first tumour is unprocessed (NT) or intravenously it is handled with MG1-GFP, the second tumour was since the 25th day, every other day with each 100 μ g of ICI(, peritonaeum It is interior) it handles or does not handle, 5 injections are carried out in total.Dotted line represents the number of days of MG1 processing.The statistical analysis of measurement of tumor: *: p < The multiple double tail t of 0.05, * *: p < 0.01, * * *: p < 0.001(non-matching are examined).Difference * table between NT and MG1+ICI group Show, the difference between MG1 and MG1+ICI group is indicated with #, and the difference between ICI and MG1+ICI group is indicated with x.For survival Curve: *: p < 0.01 *: p < 0.001(Mantel-Cox of * * of * test).

The schematic diagram for the treatment of group in Figure 19: I phase/II clinical trial phase, the clinical trial testing is with can not cure Expressing in the patients with solid tumor of MAGE-A3 (each has transgenosis using adenovirus vaccine (AdMA3) and MG1(MG1MAE3) MAGE-A3 insertion) first exempt from: reinforce the effect of strategy.B group and C group start AdMA3 in (- 14) day and are administered.

Figure 20: attached drawing show with shown dosage with AdMA3(" antibody "), MG1MA3(" MG1 ") or both processing come From in the individual tumors biopsy of the patient of the clinical test of Figure 19, times of PDL1 expression (after processing compared to processing before) Number variation.

Figure 21: attached drawing shows the mixed rumour of all dosage in A group, B group and C group in the patient of Present clinical test The multiple variation of PDL1 expression (after processing compared to processing before) in biopsy.

Specific embodiment

It has been found that the combination treatment of oncolytic virus (such as oncolytic rhabdovirus) and checkpoint inhibitor causes cancer to be controlled The unexpected for the treatment of improves.Relative to the single administration of any component, when simultaneously, when applying in order or dividually, oncolytic virus It cooperative interacts and is even synergistically interacted to significantly improve survival with checkpoint inhibitor, while without apparent secondary Effect or the reduction of virus titer.This unexpected effect can reduce the effective dose of every kind of component, to reduce pair Act on and improve the clinical effectiveness of compound and treatment.

In several embodiments, provide for treating in mammals and/or pre- anti-cancer and/or transfer are built Vertical combination treatment comprising the oncolytic virus for (i) having replication capacity and (ii) immunologic test point is co-administered to mammal The combination of inhibitor.In preferred embodiments, application has the oncolytic of replication capacity sick before immunologic test point inhibitor Poison.

Oncolytic virus

In preferred embodiments, the oncolytic virus for having replication capacity of the combination is oncolytic rhabdovirus.

Oncolytic rhabdovirus as oncolytic virus be used in combination in have the advantages that it is several, include the following: (1) for oncolytic bullet It is rare in the most of crowds of the antibody of shape virus in the world.(2) rhabdovirus is than other oncolytic virus (such as adenopathy Poison, reovirus, morbilli, parvovirus, retrovirus and HSV) replicate faster.(3) rhabdovirus grows supreme drop Degree, can be filtered by 0.2 micron filter.(4) oncolytic rhabdovirus and its recombinant have extensive host range, can feel Many different types of cancer cells are contaminated, and not by the limit of the receptor (such as Coxsack, morbilli, adenovirus) on specific cells System.(5) rhabdovirus of the invention is suitable for genetic manipulation.(6) rhabdovirus also has cytoplasm life cycle, and not whole It closes into host cell inhereditary material, this give more favorable safeties.

Typical rhabdovirus is rabies and vesicular stomatitis virus (VSV), is most study in the virus family Virus.Rhabdovirus is a kind of bullet shaped virus with non-segmentation (-) ariyoshi rna gene group.Rhabdovirus family include but Be not limited to: Arajas virus, Chandipura virus (AF128868/gi:4583436, AJ810083/gi: 57833891、AY871800 / gi:62861470、AY871799 / gi:62861468、AY871798 / gi: 62861466、AY871797 / gi:62861464、AY871796 / gi:62861462、AY871795 / gi: 62861460、AY871794 / gi:62861459、AY871793 / gi:62861457、AY871792 / gi: 62861455, AY871791/gi:62861453), Cocal viral (AF045556/gi:2865658), Isfahan virus (AJ810084/gi:57834038), Maraba virus (the SEQ ID ON:1-6 of U.S. Patent number 8,481,023, by drawing With being incorporated herein, HQ660076.1), Carajas virus (the SEQ ID ON:7-12 of U.S. Patent number 8,481,023, by drawing With being incorporated herein, AY335185/gi:33578037), Piry virus (D26175/gi:442480, Z15093/gi: 61405), vesicular stomatitis Alagoas virus, 157575 virus of BeAn, Boteke virus, Calchaqui virus, american badger Virus, Gray Lodge virus, Jurona virus, Klamath virus, Kwatta virus, La Joya virus, Malpais Spring virus, Mount Elgon bat viruses (DQ457103/gi | 91984805), Perinet virus (AY854652/ Gi:71842381), Tupaia viral (NC_007020/gi:66508427), Farmington, Bahia Grande virus (the SEQ ID ON:13-18 of U.S. Patent number 8,481,023, be incorporated herein by reference, KM205018.1), Muir Springs virus (KM204990.1), Reed Ranch virus, Hart Park virus, Flanders virus (AF523199/ gi:25140635、AF523197 / gi:25140634、AF523196 / gi:25140633、AF523195 / gi: 25140632, AF523194/gi:25140631, AH012179/gi:25140630), Kamese virus, Mosqueiro Virus, Mossuril virus, Barur virus, Fukuoka (Fukuoka) virus (AY854651/gi:71842379), Ke Enxia Paddy (Kern Canyon) virus, Nkolbisson virus, Le Dantec viral (AY854650/gi:71842377), Keuraliba virus, Connecticut virus, New Minto virus, Sawgrass virus, Chaco virus, Sena Madureira virus, Timbo virus, Almpiwar viral (AY854645/gi:71842367), Aruac virus, Bangoran virus, Bimbo virus, Bivens Arm virus, Blue crab virus, Charleville virus, Coastal Plains virus, 7292 virus of DakArK, Entamoeba virus, Garba virus, Gossas virus, Humpty Doo virus (AY854643/gi:71842363), Joinjakaka virus, Kannamangalam virus, Kolongo virus (DQ457100 / gi | 91984799 nucleoprotein (N) mRNA, part cds), Koolpinyah virus, Kotonkon virus (DQ457099/gi | 91984797, AY854638/gi:71842354), Landjia virus, Manitoba virus, Marco virus, Nasoule Virus, Navarro virus, Ngaingan viral (AY85464/gi:71842375), Oak-Vale virus (AY854670/ Gi:71842417), Obodhiang virus (DQ457098/gi | 91984795), Oita virus (AB116386/gi: 46020027), Ouango virus, Parry Creek virus (AY854647/gi:71842371), Rio Grande Cichlid virus, Sandjimba virus (DQ457102/gi | 91984803), Sigma virus (AH004209/gi: 1680545, AH004208/gi:1680544, AH004206/gi:1680542), Sripur virus, Sweetwater Branch virus, Tibrogargan virus (AY854646/gi:71842369), Xiburema are viral, Yata is viral, Rhode Island, Adelaide River virus (U10363/gi:600151, AF234998/gi:10443747, AF234534/gi:9971785, AY854635/gi:71842348), Berrimah virus (AY854636/gi: 71842350), Kimberley viral (AY854637/gi:71842352) or bovine ephemeral fever virus (NC_002526/ gi:10086561)。

In preferred embodiments, the combined oncolytic virus is wild type Maraba bacterial strain rhabdovirus or its change Allosome, optionally through gene modification, such as to improve tumor-selective.Maraba virus may, for example, be as the U.S. is special Sharp number 9,045,729(entire contents are incorporated herein by reference) the 2nd column 24-42 row described in Maraba virus, It has at 242 amino acid of G-protein and/or at 123 amino acid of M albumen replaces.In particularly preferred embodiment In, Maraba virus is if Brun et al. is in Mol. Ther., 18 (8): Maraba described in 1440-1449 (2010) MG1.Maraba MG1 is the Maraba bacterial strain rhabdovirus through gene modification, contains G-protein mutation (Q242R) and M albumen It is mutated (L123W), makes virus high toxicity in cancer cell, but decay in normal cell.

In another preferred embodiment, oncolytic rhabdovirus is VSV strain or its variant, optionally through base Because of modification, such as to improve tumor-selective.In particularly preferred embodiments, if Stojdl et al. is in Cancer Cell., 4(4): 263-75(2003) described in (its content is incorporated herein by reference), VSV is included in 51 sites of M albumen The methionine deletion at place.

In other preferred embodiments, oncolytic rhabdovirus expresses one or more tumor associated antigens, such as: cancer Embryo (oncofetal) antigen, such as alpha-fetoprotein (AFP) and carcinomebryonic antigen (carcinoembryonic antigen, CEA)； Surface glycoprotein, such as CA 125；Oncogene, such as Her2；Melanoma associated antigen, such as dopachrome tautomerase (DCT), GP100 and MART1；Cancer-testis antigen, such as MAGE albumen and NY-ESO1；Viral oncogene, for example, HPV E6 and E7；And in the tumour for being typically limited to embryo or embryo outside organization ectopic expression albumen, such as PLAC or tumour correlation be anti- Former variant.In such a situation it is preferred to which combination treatment to be applied to the people of the cancer with expression tumor associated antigen.It is swollen " variant " of tumor related antigen refers to that (a) includes at least one tumor associated antigen epitope from tumor-associated antigen protein Albumen, and (b) with tumor-associated antigen protein at least 70%, preferably at least 80%, more preferably at least 90% or at least 95% Identical albumen.In " Database of T cell-defined human tumor antigens:the 2013 Update. " in Cancer Immun 2013 13:15 and www.cancerimmunity.org/peptide, Van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B are provided and are summarized generally acknowledged epitope Database.Therefore, in various embodiments, the oncolytic rhabdovirus (such as VSVdelta51 or Maraba MG1) of the combination Amino acid of the coding comprising SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13 The albumen of sequence or at least 95% identical variant.In the relevant embodiments, the combined oncolytic rhabdovirus includes The rna form of reverse complemental and transgenosis, the transgenosis include the nucleosides of SEQ ID NO:2,3,5,6,8,9,11,12 or 14 Acid sequence.

In particularly preferred embodiments, oncolytic rhabdovirus expression MAGEA3, human papilloma virus E6/E7 merge egg White, human prostate albumen six cross-film epithelium antigens or Cancer Testis Antigens 1.It has been confirmed that in prime-boost strategy, table Increasing survival in relevant animal cancer model up to the oncolytic rhabdovirus of each in these tumor associated antigens, (WIPO is public The number of opening WO2014/127478)." prime-boost " used herein is directed to mammal application (the preferably vein with cancer It is interior) (replicability) oncolytic rhabdovirus, natural tumor associated antigen relevant to the cancer is expressed, and the lactation is dynamic Object to it with pre-existing immunity, it is wherein pre-existing in mammal to exempt to reinforce pre-existing immunity Epidemic disease power is preferably established by exempting from the beginning of the forward direction mammal of application oncolytic rhabdovirus to apply tumor associated antigen.It is preferred that Ground, mammal suffer from the cancer for having detected/having identified the expression of tumor associated antigen.

Just exempting from step (can use any suitable administration method, including but not limited to intravenously, intramuscular by application Or intranasal administration) tumor associated antigen itself, or preferably by being realized via vector administration tumor associated antigen, the carrier Such as adenovirus, poxvirus (such as vaccinia virus), retrovirus (such as slow virus) or α virus (such as semliki Forest) the antigen presenting cell (such as dendritic cells) of carrier or plasmid or loading.In mammals, make for applying The first carrier for exempting from application carried out with tumor associated antigen, is different from (i.e. heterologous in) in immunology and is administered and carrys out booster immunization The oncolytic virus of the expression tumor associated antigen of power is (for example, the oncolytic virus in expression tumor associated antigen is oncolytic bullet shape disease In the case where poison, just exempting from carrier not is rhabdovirus different in rhabdovirus or immunology).In general, in mammals, Using generally acknowledged recombinant technique modification carrier to express antigen, and to generate the effective quantity of immune response application carrier.Citing comes It says, at least about 10 is applied into mouse muscle⁷The adenovirus vector of the expression tumor associated antigen of pfu, it is sufficient to generate immune answer It answers.Treatment for people, for example, about 10 can be applied⁸-10¹²、10⁹-10¹¹Or 10¹⁰The gland of the expression tumor associated antigen of pfu Viral vectors is to generate initiation immune response.

Once in mammals by tumor associated antigen it is first exempt from apply (such as passing through adenovirus vector) produce Immune response expresses identical tumour phase then in suitable immune response interval effectively to measure application to oncolytic viral therapy At least once, the interval may, for example, be at least about 24 hours, preferably at least about 2-4 days to the oncolytic rhabdovirus of pass antigen Or it is longer, in for example, about one week, in about two weeks, in about three weeks or about in surrounding.

In some embodiments, the first time for expressing the oncolytic rhabdovirus of tumor associated antigen reinforces application and occurs The single of identical tumor associated antigen just exempts to apply after (such as passing through adenovirus vector) about two weeks, then exempts to apply at the beginning of the single Second of reinforcement application can be carried out with latter about 15-20 days, about 16-19 days or about 17 days.In relevant embodiment, checkpoint The first time dosage of inhibitor is exempted from the beginning of the single after application and in the oncolytic rhabdovirus for expressing identical tumor associated antigen First time reinforce applying before application, and it is related to identical tumour is expressed anti-to preferably include the application of checkpoint inhibitor The treatment phase of the application overlapping of former oncolytic rhabdovirus.In other embodiments, second of dosage of checkpoint inhibitor Be for the first time, second (and optionally third time, the 4th time, it is the 5th inferior) reinforce application after apply.In related embodiment party In case, weekly, every other week or every three weeks application checkpoint inhibitor.

In De Plaenet al, codes for tumor spy is discussed in Immunogenetics 40:360-369 (1994) The MAGE gene family of Specific Antigen.MAGEA3 is expressed in kinds of tumors, including melanoma, non-small cell lung cancer, head and neck cancer, Colorectal cancer and bladder cancer.The tumor associated antigen epitope of MAGEA3 is identified.Therefore, the variant of MAGEA3 albumen It can be, the antigen protein including at least one tumor associated antigen epitope selected from the following: EVDPIGHLY (SEQ ID NO: 1)、FLWGPRALV(SEQ ID NO: 2)、KVAELVHFL(SEQ ID NO: 3)、TFPDLESEF(SEQ ID NO: 4)、 VAELVHFLL(SEQ ID NO: 5)、MEVDPIGHLY(SEQ ID NO: 6)、EVDPIGHLY(SEQ ID NO: 7)、 REPVTKAEML(SEQ ID NO: 8)、AELVHFLLL(SEQ ID NO: 9)、MEVDPIGHLY(SEQ ID NO: 10)、 WQYFFPVIF(SEQ ID NO: 11)、EGDCAPEEK(SEQ ID NO: 12)、KKLLTQHFVQENYLEY(SEQ ID NO: 13)、RKVAELVHFLLLKYR(SEQ ID NO: 14)、KKLLTQHFVQENYLEY(SEQ ID NO: 15)、 ACYEFLWGPRALVETS(SEQ ID NO: 16)、RKVAELVHFLLLKYR(SEQ ID NO: 17)、VIFSKASSSLQL (SEQ ID NO: 18)、VIFSKASSSLQL(SEQ ID NO: 19)、VFGIELMEVDPIGHL(SEQ ID NO: 20)、 GDNQIMPKAGLLIIV(SEQ ID NO: 21)、TSYVKVLHHMVKISG(SEQ ID NO: 22)、 RKVAELVHFLLLKYRA (SEQ ID NO:23) and FLLLKYRAREPVTKAE (SEQ ID NO:24)；And with MAGEA3 albumen at least 70%, 80%, 90% or 95% identical albumen.The variant of tumor-associated antigen protein may need Only include the epitope with hypermorph allel frequency, is greater than 40% frequency of group.Therefore, MAGEA3 variant Preferred example may include the albumen containing at least one epitope selected from the following: FLWGPRALV (SEQ ID NO: 25)、KVAELVHFL(SEQ ID NO: 26)、EGDCAPEEK(SEQ ID NO: 27)、KKLLTQHFVQENYLEY(SEQ ID NO:28), RKVAELVHFLLLKYR (SEQ ID NO:29) and KKLLTQHFVQENYLEY (SEQ ID NO:30)；And With MAGE A3 albumen at least 70%, 80%, 90% or 95% identical albumen.

Human papilloma virus (HPV) cancer protein E6/E7 constitutive expression (Zur Hausen, H(1996) in cervical carcinoma Biochem Biophys Acta 1288:F55-F78).In addition, the reason of type of HPV 16 and 18 is 75% cervical carcinoma (Walboomers JM(1999) J Pathol 189:12-19).First exempt from heterologous: reinforcing in setting, express 16 He of HPV The oncolytic rhabdovirus (it is mutated to remove carcinogenic potential) of the fusion protein of the E6/E7 cancer protein of 18 types, has been shown Increase the number and percentage of antigen-specificity CD8+ T cell out.

Six cross-film epithelium antigens (the Six-Transmembrane Epithelial Antigen of the of prostate Prostate, huSTEAP) it is a kind of albumen found recently, it is over-expressed in carcinoma of prostate, and in kinds cancer It is raised in cell line, including pancreas, colon, mammary gland, testis, uterine neck, bladder, ovary, acute lymphoblastic, leukaemia and Juventus Sarcoma (Hubert RS et al., (1999) Proc Natl Acad Sci 96:14523-14528).STEAP gene coding There are six potential trans-membrane regions, the albumen that flank is hydrophilic amino and carboxy-terminal domains for tool.Have shown that expression The oncolytic rhabdovirus of huSTEAP first is exempted from heterologous: increasing the quantity of antigen-specificity CD8+ T cell in enhancing setting And percentage.

Cancer Testis Antigens 1(Cancer Testis Antigen 1, NYES01) it is in normal adult tissue (such as testis Ball and ovary) and the cancer/testis antigen (Nicholaou the T et al., (2006) Immunol that are expressed in various cancers Cell Biol 84:303-317).Cancer Testis Antigens are a kind of unique antigen families, are limited in normal adult pair The expression of Testicular Germ Cell, but the unconventionality expression on various entity tumors, including soft tissue sarcoma, melanoma and epithelioma. It has shown that the oncolytic rhabdovirus of expression NYES01 first is exempted from heterologous: increasing antigen-specificity CD8 in enhancing setting The number and percentage of+T cell.

In other embodiments, the oncolytic rhabdovirus for expressing tumor associated antigen and checkpoint inhibitor are applied jointly With to suffer from cancer mammal, wherein the mammal to the tumor associated antigen have naturally occurring immunity.

Therefore, it in several embodiments, provides for treating in mammals and/or the method for pre- anti-cancer, It includes that (i) expression and the natural relevant natural tumor associated antigen of cancer are co-administered to the mammal with cancer Oncolytic rhabdovirus, and mammal has pre-existing immunity, and (ii) checkpoint inhibitor to the antigen, wherein Pre-existing immunity is preferably by being applied to lactation for tumour antigen before applying oncolytic rhabdovirus in mammal Animal is established.In preferred embodiments, oncolytic rhabdovirus is applied to mammal intravascularly.It is excellent at other In the embodiment of choosing, pre-existing immunity is dynamic by the forward direction lactation in application oncolytic rhabdovirus in mammal The viral vectors (such as adenovirus) of object application expression tumor associated antigen is come what is established.

The approach of the application of the combined oncolytic virus can change with the position of lesion and property naturally, and wrap Include for example intradermal, transdermal, parenteral, intravascular (intravenous or intra-arterial), intramuscular, intranasal, subcutaneous, regional, percutaneous, gas In pipe, in peritonaeum, in bladder, in tumour, sucking, perfusion, lavation, direct injection, nutrition, oral administration and pharmaceutical formulation.? In preferred embodiment, include the combined oncolytic virus (such as oncolytic rhabdovirus) and pharmaceutically acceptable carrier Pharmaceutical composition by intratumor injection and/or it is intravascular application be administered to the mammal with cancer, although described Pharmaceutical composition each by reference can be integrally incorporated this such as 5,543,158,5,641,515 and 5,399,363(of United States Patent (USP) Text) described in, alternatively by tumour, it is parenteral, intravenous, intra-arterial, intradermal, intramuscular, transdermal or even in peritonaeum Application.As used herein, " carrier " includes any and all solvents, decentralized medium, carrier, coating, diluent, antibacterium and resists Epiphyte pharmaceutical, etc. blend absorption delaying agent, buffer, carrier solution, suspension, colloid etc..These media and medicament are used for drug The purposes of active material is well known in the art.Unless any conventional media or reagent are incompatible with active constituent, otherwise consider Its purposes in therapeutic combination.The active constituent of supplement can also mix in composition.

In certain embodiments, tumour to be treated at least initially may not be resectable.Use therapeutic virus The treatment that construct carries out can increase the resectability of tumour, this is because the contraction of edge or certain special by eliminating Invasive part.After treatment, resection is possible.Other treatment after excision will be used to eliminate the microscopic residual of tumor locus Disease.

For tumor bed after primary tumor or excision, typical therapeutic process will be related to multi-dose.Typical primary Oncotherapy includes 1,2,3,4,5,6 or more dosage of application at 1,2,3,4,5,6 week or in the longer time.Two weeks schemes can To be repeated once, twice, three times, four times, five times, six times or more times.Over the course for the treatment of, completion plan can be reappraised Dosage needs.

Treatment may include various " unit dose ".Unit dose is defined as the therapeutic combination containing predetermined amount.Wait apply Amount and specific approach and preparation, in the technical scope of the personnel of clinical field.Unit dose is not needed as list Secondary injection application, but may include the continuous infusion within a period of time of setting.Unit dose of the invention can be in virus It is easily described in terms of the plaque forming unit (pfu) or virion of construct.The range of unit dose is 10³、10⁴、 10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³Pfu or vp and higher.Alternatively, according to the type of virus and can reach Titre, can be by 1 to 100,10 to 50,100-1000 or up to about 1 × 10⁴、1×10⁵、1×10⁶、1×10⁷、1×10⁸、 1×10⁹、1×10¹⁰、1×10¹¹、×10¹²、1×10¹³、1×10¹⁴Or 1 × 10¹⁵Or higher infectious viral particle (vp) It is delivered to the cell of patient or patient.

Phrase " pharmaceutically acceptable " or " pharmacologically acceptable " refer to do not generated when being applied to people allergy or The molecular entity and composition of similar adverse reaction.Contain preparation of the albumen as the Aquo-composition of active constituent in ability It is known in domain.In general, injection is made in this composition, liquid solution or suspension can be, can also be prepared as Suitable for the solid form being dissolved or suspended in liquid before the injection.

Checkpoint inhibitor

Immunologic test point adjusts the T cell function in immune system.T cell it is cell-mediated it is immune in play an important role.Inspection It makes an inventory of albumen and specific ligand interacts, signal is sent into T cell and closes or inhibit T cell function by these ligands. Cancer cell utilizes this point by driving the high level expression of the checkpoint albumen on its surface in turn, to control T Cell expresses the checkpoint albumen into tumor microenvironment on the surface of T cell, to inhibit anticancer immune response.

It is any compound for inhibiting immunologic test point protein function for the combined immunologic test point inhibitor.Suppression System includes that function is reduced and retardance completely.Particularly, immunologic test point albumen is people's immunologic test point albumen.Therefore, inspection is immunized Make an inventory of the inhibitor that inhibitor is preferably people's immunologic test point albumen.Art describes immunologic test point albumen (see, for example, Pardoll, Nature Rev. Cancer 12(4): 252-264 (2012)。

Checkpoint albumen include but is not limited to CTLA4, PD-1 and its ligand PD-L1 and PD-L2, B7-H3, B7-H4, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, TIGIT and BTLA.It is related to LAG-3, BTLA, B7H3, B7H4, TIM3 and KIR Approach be considered to constitute in the art similar to CTLA-4 and PD-1 Dependent immunologic test point approach (see, for example, Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480-489)。

Preferred immunologic test point protein inhibitor is the antibody for specifically identifying immunologic test point albumen, preferably people or Humanized monoclonal antibodies.Have been described many CTLA-4, PD1, PDL-1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, TIGIT and KIR inhibitor.

The checkpoint CTLA-4 inhibitor includes but is not limited to a kind of complete people CTLA-4 blocking antibody of ipilimumab(, mesh The preceding title with Yervoy (Bristol-Myers Squibb) is sold), tremelimumab(referring to Ribas et al., J. (2013) Clin. Oncol. 31:616-622), in U.S. Patent Application Publication No. 2005/0201994,2002/ Antibody disclosed in 0039581 and 2002/086014 (its respective content is incorporated herein by reference) and in United States Patent (USP) Numbers 5,811,097,5,855,887,6,051,227,6,984,720,6,682,736,6,207,156,5,977,318,6, 682,736, antibody disclosed in 7,109,003 and 7,132,281 (its respective content is incorporated herein by reference).

PD-1 inhibitor includes but is not limited to the humanized antibody for blocking people PD-1, such as lambrolizumab(for example exists U.S. Patent number 8,354,509(is incorporated herein by reference) in and in Hamid et al., N. Engl. J. Med. HPD109A disclosed in 369:134-144 (2013) and its humanized derivative thereof h409A11, h409A16 and h409A17), Pidilizumab(CT-011；It is public in Rosenblatt et al., J Immunother. 34:409-418 (2011) Open) and fully human antibodies (fully human antibody), such as nivolumab(CAS registration number: 946414-94-4； It is formerly referred to as MDX-1106 or BMS-936558, Topalian et al., N. Eng. J. Med. 366:2443-2454 (2012), disclose, be incorporated herein by reference in U.S. Patent number 8,008,449) or weight comprising any of these antibody The antibody of chain and light chain variable region.Pidilizumab is a kind of complete people IgG4 monoclonal antibody, in human clinical trial The effect of showing treatment diffusivity large B cell lymphoid tumor.Nivolumab is a kind of complete people IgG4 monoclonal antibody, has been shown The therapeutic efficiency for the treatment of of late stage Refractory Malignant Tumor (such as melanoma, clear-cell carcinoma and NSCLC) is shown.Other PD-1 inhibit Agent may include fusion protein, such as PD-L2-Fc fusion protein, and also referred to as B7-DC-Ig or AMP-244(are disclosed in Mkrtichyan M, et al. J Immunol. 189:2338-47 2012).AMP224 is suffering from pair of advanced cancer The test of I phase is carried out as monotherapy in the treatment of elephant.

In preferred embodiments, immunologic test point inhibitor is nivolumab or comprising containing nivolumab The heavy chain variable region of heavy chain variable amino acid sequence and/or chain variable region amino acid sequence containing nivolumab it is light The anti-PD-1 antibody of the separation of chain variable region.The sequence of heavy chain of nivolumab is:

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRD NSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:31)。

The sequence of light chain of nivolumab is:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTL TISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID NO: 32)。

In some preferred embodiments, checkpoint inhibitor includes the heavy chain and/or sequence of light chain with nivolumab At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 98%, At least 99% or 100% identical heavy chain and/or sequence of light chain.

Immunologic test point inhibitor further includes but is not limited to block the humanization or fully human antibodies of PD-L1, such as Pembrolizumab(CAS registration number 1374853-91-4；Also referred to as MK-3475) (being disclosed in WO2009/114335), MEDI-4736(is disclosed in U.S. Patent number 8,217,149, is incorporated herein by reference), MPDL33280A(is in United States Patent (USP) Disclosed in numbers 8,217,149, content is incorporated herein by reference), MIH1(Affymetrix, can be obtained by eBioscience (16.5983.82)), BMS-936559 and MSB0010718C (Avelumab) or heavy chain comprising any of these antibody and light The antibody of chain variable region.BMS-936559 is a kind of complete people IgG4 monoclonal antibody, proved in the clinical test of people its It effectively (is applied every two weeks primary) in terms for the treatment of melanoma, NSCLC, clear-cell carcinoma and ovarian cancer.Pembrolizumab is one Kind humanization IgG4 monoclonal antibody, changes with stable SER228PRO sequence in the region Fc, is carrying out clinical examination It tests, for therapeutic advance type, Locally Advanced or metastatic cancer, melanoma or NSCLC, is bound to PD-1 and prevents The interaction of PD-1 and its ligand PD-L1 and PD-L2.MPDL33280A is a kind of monoclonal antibody, is suffering from BRAF V600 It is mutated in the object of metastatic melanoma to combine with BRAF inhibitor vemurafenib and be tested, and with evening It combines and is tested with the bevacizumab of targeting VEGFR in the object of phase solid tumor.MEDI-4736 is in the evil for suffering from advanced stage Property melanoma, clear-cell carcinoma, NSCLC and colorectal cancer patient in carry out Phase I clinical trial.

In particularly preferred embodiments, immunologic test point inhibitor is pembrolizumab or comprising containing The heavy chain variable region of the heavy chain variable amino acid sequence of pembrolizumab and/or light chain containing pembrolizumab can Become the anti-PD-1 antibody of the separation of the light chain variable region of region amino acid sequence.The sequence of heavy chain of pembrolizumab is:

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTD SSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 33)。

The sequence of light chain of pembrolizumab is:

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGT DFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 34)。

In some preferred embodiments, checkpoint inhibitor includes the heavy chain and/or light chain with pembrolizumab Sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, At least 98%, at least 99% or 100% identical heavy chain and/or sequence of light chain.

In preferred embodiments, the combined immunologic test point inhibitor presses down selected from CTLA-4, PD-1 or PD-L1 Preparation, such as, but not limited to: pembrolizumab, ipilimumab, tremelimumab, labrolizumab, Nivolumab, pidilizumab, AMP-244, MEDI-4736, MPDL33280A or MIH1.These immunologic tests can be used The known inhibitor of point albumen, or analog can be used, especially chimeric, humanization or person form antibody.

As it is known by the man skilled in the art, substitution and/or equivalent title can be used for above-mentioned certain antibody.These are replaced Generation and/or equivalent title is interchangeable in the context of the present invention.For example, as it is known that the substitution of lambrolizumab And equivalent title MK-3475 and pembrolizumab.

Other immunologic test point inhibitor of the combination include but is not limited to target immunologic test point albumen and be related to PD- The reagent of the approach of L2, LAG3, BTLA, B7H4, TIM3 and TIGIT.For example, people PD-L2 inhibitor known in the art includes MIH18(is described in Pfistershammer et al., Eur J Immunol. 36:1104-1113 (2006)).This field Known LAG3 inhibitor includes solubility LAG3(IMP321 or LAG3-Ig, is disclosed in U.S. Patent Application Publication No. 2011-0008331(is incorporated herein by reference) and Brignon et al., Clin. Cancer Res. 15:6225- 6231 (2009)) and block mouse or humanized antibody (such as IMP701 and the U.S. Patent Application Publication No. of people LAG3 2010-0233183 description other antibody, be incorporated herein by reference), or block people LAG3 fully human antibodies (such as Antibody disclosed in BMS-986016 and U.S. Patent Application Publication No. 2011-0150892, is incorporated herein by reference).

The combined BTLA inhibitor include but is not limited to block people BTLA and its ligand interaction antibody (such as 4C7 disclosed in U.S. Patent number 8,563,694, is incorporated herein by reference).

The checkpoint B7H4 inhibitor include but is not limited to be directed to people B7H4 antibody (be disclosed in WO2013025779 A1 and U.S. Patent Application Publication No. 2014/0294861, is incorporated herein by reference) or B7H4 Soluble recombinant versions (such as beauty Disclosed in state's patent application publication number 2012/0177645, it is incorporated herein by reference or anti-human B7H4 clones H74: EBiocience # 14-5948).

The checkpoint B7-H3 inhibitor includes but is not limited to neutralize the antibody of human B 7-H 3 (for example, U.S. Patent Application Publication MGA271 disclosed in number 2012/0294796 as BRCA84D and derivative, is incorporated herein by reference).

The checkpoint TIM3 inhibitor includes but is not limited to target the antibody of people TIM3 (for example, being such as incorporated herein by reference U.S. Patent number 8, disclosed in 841,418, or by Jones et al., J Exp Med., 205 (12): 2763- Anti-human TIM3, blocking antibody F38-2E2 disclosed in 79 (2008)).The checkpoint KIR inhibitor includes but is not limited to Lirilumab(is described in Romagne et al., Blood, 114 (13): 2667-2677 (2009)).Known immune inspection The inhibitor for making an inventory of albumen can be used or be can be used the antibody of analog, especially chimeric versions thereof with its form known, most It is preferred that humanization form.The checkpoint TIGIT inhibitor preferably inhibits the phase interaction of TIGIT and polovirus receptor (CD155) With including but not limited to antibody (such as U.S. Patent number 9,499,596 and the U.S. Patent Application Publication No. of targeting people TIGIT 20160355589, disclosed in 20160176963 those) and polovirus variant (such as in U.S. Patent number 9,327, Disclosed in 014 those).

In some respects, combination as described herein includes more than one the immune inspections of (i) within each aspect of the present invention Make an inventory of inhibitor and (ii) oncolytic virus.Preferably, more than one described immunologic test point inhibitor be selected from CTLA-4, PD-1 or PD-L1 inhibitor.For example, ipilimumab(anti-CTLA 4) verified face with treatment while the anti-PD1 of Nivolumab() Bed activity seem be different from monotherapy in obtain clinical activity (Wolchok et al., N. Eng. J. Med., 369:122-33 (2013)).Other examples include the checkpoint LAG3 inhibitor and the anti-checkpoint PD-1 inhibitor (Woo et Al., (2012) Cancer Res. 72:917-27) or the checkpoint LAG3 inhibitor and the checkpoint PD-L1 inhibitor (Butler et al., Nat. Immunol., 13:188-195 (2011)).

In other respects, combination as described herein including (i) one or more checkpoint inhibitor and has proven to improve institute The one or more additional therapeutic agents for the effect of stating one or more checkpoint inhibitor, and (ii) oncolytic virus.For example, Lirilumab(is also referred to as anti-KIR, BMS-986015 or IPH2102, as disclosed in United States Patent (USP) No.8119775) and easily Puli's nurse Ma (ipilimumab) (clinicaltrials.gov NCT01750580) combination or with receive military monoclonal antibody (nivolumab) (clinicaltrials.gov NCT01714739) is combined.Another example be target ICOS medicament and The checkpoint CTLA-4 inhibitor (Fu et al., Cancer Res., 71:5445-54 (2011), or the medicine of targeting 4-1BB Agent (such as urea) and the checkpoint CTLA-4 inhibitor (Curran et al., PloS 6 (4): 9499 (2011)).Other examples Attached bag includes the checkpoint PD-1/PD-L1 inhibitor and pazopanib (pazopanib), Sutent (sunitinib), replaces up to sand Buddhist nun (dasatinib), INCR024360, PegIFN-2b, Erlotinib (Tarceva), examine than for Buddhist nun (Cobimetinib) and/or Trimetinib (Trametinib), Debrafinib.In some preferred embodiments, the combination includes oncolytic bullet shape disease Poison and the Puli nurse Ma of military monoclonal antibody+pazopanib/Sutent/easily of (i) receiving, (ii) receive military monoclonal antibody+Dasatinib, (iii) Pyridine aldoxime methyliodide (PAM) monoclonal antibody+INCR024360, (iv) pyridine aldoxime methyliodide (PAM) monoclonal antibody+pazopanib, (v) pyridine aldoxime methyliodide (PAM) monoclonal antibody+PegIFN-2b, (vi) MED14736+dabrafenib (Dabrafenib)/Trimetinib, (vii) MPDL3280A+Erlotinib or (viii) MPDL3280A+examine than for Buddhist nun.

Checkpoint inhibitor disclosed herein can be applied by all means, comprising: such as oral and extra-parenteral administration, Such as intravenous, intramuscular, subcutaneous, socket of the eye is interior, in intracapsular, peritonaeum, rectum is interior, in brain pond, tumour it is interior, intravascular, intradermal or logical It crosses skin passively or promotes to absorb, such as use skin patch or transdermal iontophoretic therapy respectively.Checkpoint inhibitor It can be applied to the position of pathological state, for example, intravenous or intra-arterial enters the blood vessel of supply tumour.

The total amount for the medicament applied when practicing method of the invention can be used as single dose and be applied to object, can be logical It crosses within the relatively short time and injects or be transfused to object, or fractionated therapy plan can be used and be applied to object, wherein Multi-dose is applied in longer time section.One skilled in the art will appreciate that the amount of the composition of the pathological condition in treatment object Depending on many factors, the number of the treatment at age and general health and administration method and application including object.Mirror In these factors, those skilled in the art will adjust given dose as needed.In general, initially use I phase and the examination of II phase clinic Test the preparation of determining composition and the approach of application and frequency.

In certain embodiments, checkpoint inhibitor is with 0.01-0.05 mg/kg, 0.05-0.1 mg/kg, 0.1-0.2 mg/kg、0.2-0.3 mg/kg、0.3-0.5 mg/kg、0.5-0.7 mg/kg、0.7-1 mg/kg、1-2 mg/kg、2-3 mg/ Kg, 3-4 mg/kg, 4-5 mg/kg, 5-6 mg/kg, 6-7 mg/kg, 7-8 mg/kg, 8-9 mg/kg, 9-10 mg/kg, extremely Few 10 mg/kg, or any combination thereof dosage application.In certain embodiments, checkpoint inhibitor applies weekly at least one It is secondary, apply weekly apply at least twice, weekly apply at least three times, every two weeks at least once, every three weeks application at least once or Person monthly applies at least once or multiple months apply at least once.In the relevant embodiments, checkpoint inhibitor is applied weekly Once, application in primary, every three weeks is applied every other week once or is administered once a month.In certain embodiments, checkpoint presses down Preparation is with the application of single dosage, two dosage, three dosage, four dosage, five dosage or six dosage or more dosage. In preferred embodiments, checkpoint inhibitor is pyridine aldoxime methyliodide (PAM) monoclonal antibody, and with scheme (the preferably intravenous infusion of 2 mg/kg 30 minutes) it is administered once every three weeks.

Embodiment

Providing following embodiments is to be not meant to limit in any way in order to illustrate various embodiments of the invention The system present invention.The person skilled in the art will easily understand the present invention is perfectly suitable for realizing mentioned by the target and acquisition Objects and advantages and those of intrinsic target, objects and advantages herein.The embodiment of the present invention and side as described herein Method represents preferred embodiment at present, is exemplary, it is no intended to limit the scope of the invention.Those skilled in the art will Will recognize that include variation therein and other purposes within the spirit of the invention limited by scope of the claims.

Embodiment 1

Oncolytic rhabdovirus+checkpoint inhibitor

In the clinically relevant homogenic tumor model of immunocompetence, checkpoint inhibitor and oncolytic bullet shape disease is co-administered in assessment The effect of poison.

Material and method

To BALB/c mouse subcutaneous transplantation 5 × 10⁵A CT26(colon cancer) cell.Allow tumour growth until they reach about 250 mm³.Mouse is divided to one group (table 1) into 4 groups at random, it is ensured that the average value and variance of tumour are equal:

Table 1

。

MG1/GFP is a kind of Maraba bacterial strain rhabdovirus through gene modification, contains G-protein mutation (Q242R) and M Protein mutation (L123W) simultaneously expresses heterologous protein GFP(green fluorescent protein), on day 1 with the 3rd day with 2 × 10⁸A plaque shape It is intravenously applied at the dosage of unit (PFUs), and at the 5th day with 5 × 10⁸The dosage of PFU is intravenously applied.With every three days The dosage of 100 μ g applies anti-CTLA 4 monoclonal antibody (the Clone 9D9 in mouse source by intraperitoneal injection; BioXCell Cat. No. BE0164).Fig. 1 describes co-administration protocols.By calliper to measure, 3 days record tumours are surveyed weekly Magnitude.Gross tumor volume: 4/3 * π * L/2 * (W/2) is calculated using following formula², wherein L=length, W=width.Record institute There is the survival of mouse.Once tumour is greater than 1500 mm³, it is considered as mouse and reaches home.

The 10th day progress immunoassay after the first time dosage of MG1/GFP.Human peripheral blood is stimulated again by vitro peptide Monocyte (PBMC) completes immunoassay, and is dyed to one group of cell factor thin to assess the T of CT26 AH1- specificity The quantity of born of the same parents and measurement are multi-functional.It is assessed by being quantified to the IFN-γ list positive and the double positives of IFN-γ/TNF-α It is multi-functional.Antibody for flow cytometry comes from BD Biosciences: IFN γ-APC Cat#554413, TNF γ- FITC Cat#554418, CD107a-PE Cat#558661 come from eBiosciences:CD8-Alexa700 Cat#56- 0081-82,CD4-PerCp-Cy5.5 Cat#45-0042-82.Peptide for stimulating again comes from Biomer Technology: CT26 AH1-SPSYVYHQF,VSV/MG1 N-MPYLIDFGL.In brief, thin in the T of the 10th day measurement CT26- specificity Born of the same parents' response.Peripheral blood mononuclear cells incubates in complete RPMI with CT26 AH1 peptide, for carrying out the CD8+ of CT26- specificity T- cell (again) stimulation.Incubator (37 DEG C, 5% CO₂, 95% humidity) and middle culture 40 minutes 5 hours, in last 4 small periods Between incubated with brefeldin A (1 μ g/ml).With the antibody treated cells of targeting CD16/CD32, targeting T is then used The fluorescent labeled antibody of cell surface marker object is dyed.Then, cell carry out permeabilization and fixation and to cell within a cell because Son dyeing.Data are obtained using FACSCanto flow cytometer.

As a result

Antitumor reaction.The co-administration of anti-CTLA 4 antibody and MG1/GFP cause anti-CT26 immune response to increase.Fig. 2 explanation For every group in four groups of mouse, the CD8+ T cell of expression IFN-γ accounts for the percentage of sum when response CT26 antigen.Fig. 3 When illustrating to reply CT26 antigen respectively with Fig. 4, only secretion of gamma-IFN (single positive, to exclude the cell for also expressing TNF-α) and secretion The percentage of IFN-γ and the CD8+ T cell of TNF α (double positives exclude the cell for only expressing IFN-γ).Fig. 2-4 shows will Checkpoint inhibitor and oncolytic rhabdovirus co-administration are increased to the CD8+ T cell of immundominance CT26 antigen-specific Percentage.

Tumor size.Tumour in control-animal (control group, Fig. 5) reaches 2,000 mm in the 15th day mean size³.It is single Private anti-CTLA 4 antibody processing does not slow down tumour growth (CLTA4, Fig. 5).It is individually handled with MG1/GFP and slows down tumour life It is long, but by the 22nd day, the tumour mean size in all mouse reached 1800 mm³(MG1/GFP, Fig. 5).Use MG1/GFP Combined treatment with CTLA4 inhibitor for tumour growth statistically better than control group, individually anti-CTLA-4 and MG-1/GFP group, and during entire assessment, in the animal that the combination using MG1/GFP and CTLA4 is treated, tumour It is less than 1500 mm³(MG1/GFP+CTLA4, Fig. 5).

Survival analysis.Analyze the survival rate of the animal from each processing group.Data are as Kaplan-Meier curve It is shown in Fig. 6.The scheme that MG1/GFP is combined with anti-CTLA 4 antibody be statistically better than being used alone any medicament or (Log-rank Mantel-Cox is examined for the treatment of control group；Compared with individual MG1/GFP, 0.0051) combined p value is. The median of time-to-live is 8 days (control group), 10 days (individual anti-CTLA 4), 18 days (individual MG1/GFP) and 29 days (combination).There is the 47th day that 4 survivals to research terminates in 9 mouse in combined therapy group.On the contrary, MG1/ is administered alone Mouse in the group of GFP did not survived to the 22nd day.

Compared with any monotherapy, checkpoint inhibitor (anti-CTLA-4) and oncolytic rhabdovirus (MG1/GFP) Significant delay tumour growth is treated in combination, and compared with individual any medicament, is observed significantly using combined therapy Survival benefit.

Embodiment 2

Checkpoint inhibitor+oncolytic rhabdovirus prime-boost

In clinically relevant homogenic B16 Lung metastases model, has evaluated and checkpoint inhibitor (anti-PD-1 antibody) is co-administered Maraba rhabdovirus with expression tumour antigen is (after carrying out just exempting from application using identical tumour antigen, such as Polet al, described in (2014) Mol Ther 22 (2): 420-429, entire contents are incorporated herein by reference) to antitumor The influence of immune response.

Materials and methods.To transplanting 2.5 × 10 in C57BL/6 mouse vein⁵A B16F10 mouse melanin tumor cell, makes Tumor inoculation 5 days.One group (table 2) mouse being assigned in 4 groups.

Table 2

。

Ad-hDCT is a kind of replication-defective adenoviral (E1/E3- missing) based on human serotype 5, is modified and carrys out table Intelligent's dopachrome tautomerase (hDCT) transgenosis, with 2 × 10⁸The dosage intramuscular of pfu is applied.MG1-hDCT be by It is transformed to express the MG1 Maraba virus of hDCT transgenosis, with 1 × 10⁹The dosage of pfu is intravenously applied.Anti- PD-1 antibody ((BioXCell Cat. No. BE0146) is administered with the dosage of 250 μ g by intraperitoneal injection, 3 days weekly, continues 5 Week.The graphical overview of therapeutic scheme is as shown in Figure 7.

In the 14th day (after just exempting from) and the 20th day (expected peak value reinforcement) and the 27th day progress immunoassay.By from Body peptide stimulates again completes immunoassay on PBMC, and is dyed to one group of cell factor thin to assess the T of DCT- specificity The amount of born of the same parents and determining multifunctionality.By the double positive and IFN-γ/TNF-α of, IFN-γ/TNF-α positive to IFN-γ list/ Tri- positive cell of IL-2 is quantified to assess multifunctionality.The dyeing of CD107a marker detects CD8+ T by measurement threshing The dissolved cell activity of cell, threshing are cytolytic prerequisites.Antibody for flow cytometry comes from BD Biosciences:IFN- γ-APC Cat#554413, TNF α-FITC Cat#554418, IL-2-BV421 Cat# 562969, CD107a-PE Cat#558661 or from eBiosciences:CD8-Alexa700 Cat#56-0081-82, CD4-PerCp-CY5.5 Cat#45-0042-82.By peripheral blood mononuclear cells and SVY peptide (corresponding to being bound to H-2K^b's DCT(DCT_180-188) immunodominant epitopes, 2 μ g/ml) incubated together in complete RPMI, the CD8+ for DCT- specificity T cell (again) stimulation.Incubator (37 DEG C, 5%CO₂, 95% humidity) and middle incubation 40 minutes 5 hours, it is used in last 4 hours Brefeldin A (1 μ g/ml) is incubated.With the antibody treated cells of targeting CD16/CD32, targeting T-cells are then used The antibody of the fluorescent marker of surface marker is dyed.Then, by cell carry out permeabilization and fixation and to cell within a cell because Son dyeing.Data are obtained using FACSCanto flow cytometer.

Record the survival rate of all mouse.If showing serious respiratory distress, then it is assumed that mouse is reached home.

As a result.Exempt from time to peak point (the 14th day) just, the peptide stimulation of peripheral blood is (with identification IFN-γ, TNF-α and IL-2 Antibody dyeing) carry out 5 hours after forty minutes, intracellular cytokine dye (ICS) display: for be treated in combination with each list Solely treatment is compared, and the percentage of the CD8+ T cell of following cell factor dyeing increases: IFN-γ (single positive), and IFN-γ+ TNF-α (double positives) and IFN-γ+TNF-α+IL-2(three are positive).As a result as shown in Fig. 8 A-F.It can from Fig. 8 A-8B Out, compared with other treatment group, lead to the hundred of CD8+ T cell with the combined therapy of checkpoint inhibitor and oncolytic rhabdovirus Divide than increasing.The CD8+ T cell that individually being handled with checkpoint inhibitor does not influence to express IFN-γ (including also expresses TNF-α And/or those of IL-2) total global percentage, or only express IFN-γ and (exclude also to express TNF-α and/or IL-2 Cell) or expression IFN-γ and TNF-α or expression IFN-γ, the CD8+ T cell of TNF-α and IL-2 percentage (Fig. 8 C-8F, Compare " PD1 " group and " control " group).Compared with only being handled with the oncolytic rhabdovirus of expression tumour antigen, tumour antigen is expressed Oncolytic rhabdovirus and checkpoint inhibitor combined therapy (after applying identical tumour antigen and just exempt from), Total global percentage (Fig. 8 C), the single positive (IFN-γ) the CD8+ T for dramatically increasing the CD8+ T cell of expression IFN-γ are thin The percentage (Fig. 8 D) of born of the same parents, the percentage (Fig. 8 E) of double positive (IFN-γ+TNF α) CD8+ T cells and three positives (IFN-γ+ TNF α+IL-2) CD8+ T cell percentage (Fig. 8 F) (Fig. 8 C-8F；Compare " just exempt from: reinforcing PD1 " group and " just exempts from: adding Group by force ").

It is proved using being dyed to the ICS for reinforcing the peripheral blood that time point (the 20th day) collected in peak value for the same terms, phase For single therapy group (" PD1 " or " just exempt from: reinforcing "), in combined therapy group (" just exempt from: reinforcing PD1 ") in blood The frequency and quantity of CD8+ T cell statistically dramatically increase.Referring to Fig. 9 A-9B.At same time point, at the beginning of individually Exempt from/reinforce or anti-PD-1 treatment is compared, the CD8+ T cell sum of the generation IFN-γ of DCT- specificity is significant after combined therapy Increase (Fig. 9 D).The addition of PD-1 also results in the T cell of higher-quality DCT specificity --- the double positive (figures of IFN-γ/TNF α 10B) and IFN-γ/tri- positive cell of TNF-α/IL-2 (Figure 10 C)) dramatically increase.When assessing CD8 frequency, DCT- is special The T cell of property does not have difference (Fig. 9 A)；However, the amplification that PD-1 group is combined the enhancing in middle CD8+ T cell library causes DCT- to react The quantity of the CD8+ T cell of property dramatically increases.

It is dyed using ICS of the identical condition to the peripheral blood collected reinforcement time point (the 27th day of research) below Prove, with just exempt from/reinforcement group compared with, group is combined in middle blood CD8+ T cell frequency increase (Figure 11 A), but is not that CD8+ T is thin The quantity of born of the same parents increases (Figure 11 B).The T cell for generating IFN-γ at the time point does not have difference (Figure 11 C-11D).In the time Point, single, double and three positive CD8+ T cells frequency or quantity between any group do not have statistically-significant difference (Figure 12 A- F).

Object survival is analyzed.Data are shown in Figure 13 as Kaplan-Meier curve.The side of Ad-hDCT Case: MG1 hDCT and anti-PD-1 antibody combination are statistically significantly better than and any medicament or control group treatment are used alone (Log-rank Mantel-Cox is examined, and just exempts from/strengthening phase ratio with being used alone, 0.0388) composition p value is.Time-to-live Median be 20 days (" control group "), 20 days (independent anti-PD-1(" PD1 ")) and 67 days (" just exempt from/reinforce ").It is tied to research Beam (the 80th day), 8 in 9 animals that group is combined in (" just exempt from: reinforcing PD1 ") are not up to terminal, therefore should without calculating The median of the time-to-live of group.

Compared with individual prime-boost group, have evaluated just exempting from apply hDCT(prime-boost) after anti-PD-1 and Express influence of the combined therapy of the MG1 Maraba rhabdovirus of hDCT to mouse weight.It can be seen that phase from Figure 14 A-14C For individual prime-boost group, applying anti-PD-1 antibody does not influence the weight of mouse, no matter antibody whether with just exempt from simultaneously With single dose (ad-hDCT application) (Figure 14 A), just exempts from latter 3 days with single dose (Figure 14 B) or start within 3 days after just exempting from continuous more Dosage (Figure 14 C) is given.Therefore, regardless of application program, the toxicity of combined therapy is all not more than individual prime-boost.

Compared with individual prime-boost, have evaluated and just exempt to apply hDCT(prime-boost) anti-PD-1 and expression later Influence of the combined therapy of the MG1 Maraba rhabdovirus of hDCT for Maraba virus titer.As can be seen from Figure 15, Applying anti-PD-1 antibody will not have a negative impact to the delivering of oncolytic virus.

Checkpoint inhibitor (anti-PD-1) is added in tumour-specificity CD8+ T cell frequency and B16 tumor animal Ad-DCT is changed in terms of quality just to exempt from.It adds anti-PD-1 and also enhances Maraba-DCT reinforcement, such as tumour-specificity CD8+ T cell counts (twice of the T cell of about Ag- specificity).Importantly, with individually just exempting from/reinforcing or anti- PD-1 treatment is compared, these beneficial effects of combined therapy are related to dramatically increasing for survival rate.Combined therapy is not observed Toxic side effect, combined therapy also do not have a negative impact to the transmitting of oncolytic virus.

Embodiment 3

In triple negative breast cancer model, the effect of combination of MG1 and immunologic test point inhibitor greatly improves

Background

Triple negative breast cancer is a kind of aggressive systemic disease, and available treatment is limited.Triple negative breast cancer (TNBC) It is negative to the expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2, therefore conventional endocrine is controlled Treating (tamoxifen and Herceptin including being usually used in hormone-sensitive breast cancer) is refractory (Hudis, C. A. & Gianni, L. Triple-negative breast cancer: an unmet medical need. Oncologist 16 Suppl 1,1-11 (2011)), and the propagation property of advanced stage form makes to treat further complication.To chemotherapy resistance The patient of form lacks selection, is pushing the fast development of alternative strategy.

Use the rhabdovirus Maraba MG1 of clinical test candidate, it was demonstrated that the weight that this immune response treats TNBC The property wanted.The development of clinically relevant model is described, wherein in operation excision after the primary tumor treated, with tumour in situ Animal is excited again.In order to simulate disease palindromia in the clinically relevant environment of TNBC, describes mouse and force recurrence model Development, wherein in operation excision and handling primary tumor with MG1 before being implanted into new tumour.The virus induction effectively swells Tumor-specificity immune response simultaneously raises immunocyte into tumour.Importantly, being handled with MG1 causes tumour cell to lure PDL1 is led, and finds a greater amount of active regulatory T cells in tumour.

Method

Cell line and culture.Vero renal epithelial cell, 4T1 and EMT6 Mouse mammary cells system are purchased from American Type Culture Collection(Manassas, VA).Cell is maintained and is supplemented with 10% fetal calf serum (FBS) (Sigma Life science, St-Louis, MO) Dulbecco improvement Eagle culture medium (DMEM) (Corning cellgro, Manassas, VA) in, and in 37 DEG C, 5%CO₂Lower culture.

Virus generates and quantization.It has been previously described amplification and purifying (Brun, the J. of MG1-GFPet al. Identification of genetically modified Maraba virus as an oncolytic rhabdovirus. Mol. Ther.18, 1440–9 (2010)).In brief, with 0.01 MOI vero cells infection 24 Hour, it then harvests, filters (0.22 μm of bottle top filter (Millipore, MA, USA)) and be centrifuged culture supernatant (30100g, 90 minutes).By precipitating be resuspended in Dulbecco phosphate buffered saline (PBS) (DPBS) (Corning cellgro, Manassas, VA) in and be stored in -80 DEG C.Virus titer is determined by plaque measurement.In brief, by the sample of serial dilution Product are transferred to Vero cell monolayer, incubate 1 hour, then with the 0.5% agarose/DMEM covering for being supplemented with 10%FBS.24 is small When after count plaque.As previously described, in some experiments, using Spectrolinker XL-1000 UV crosslinking agent, make Virus is by being exposed to 120mJ/cm²Radiate 2 minutes (Zhang, J.et al. Maraba MG1 virus enhances natural killer cell function via conventional dendritic cells to reduce postoperative metastatic disease. Mol. Ther. 22, 1320–32 (2014))。

Microarray analysis.MG1-GFP with MG1-GFP or via radiation is with the single layer of 3 MOI processing 4T1 or EMT6 cell 24 hours.It collects culture supernatants and is used for CBA and elisa assay, and use RNeasy RNA extracts kit (Qiagen) RNA is extracted from cell.In MoGene2.0-st Affymetrix press-on-a-chip and analyze duplicate total serum IgE sample.It uses Transcriptome Analysis Console v3.0(Affymetrix) software analysis original document.It is further with R language Handle standardized transcript expression value.Hotspot graph is made using R LISP program LISP packet " pheatmap " v1.0.8.Using online EnrichR tool (PMID 27141961) carries out the enrichment analysis of GO function.Gene of the selection for being enriched with analysis is MG1 infection The subset of at least 4 times of gene is raised relative to non-infected cells.

Flow cytometry.Splenocyte (Roy, D. G. processed as described aboveet al. Programmable insect cell carriers for systemic delivery of integrated cancer biotherapy.J. Control. Release220, 210–221 (2015)).In brief, it collects spleen and passes through 70 μm of filters (Fisher Scientific, Waltham, MA) is smashed to pieces, is then used ACK lysis buffer splitting erythrocyte and is resuspended In FACS buffer solution (PBS, 3%FBS).Tumour cell is extracted, we use mouse tumor mixture (Miltenyi), Mild MACS pipe and mild MACS separator (Miltenyi) are used according to the scheme of manufacturer.Using CD45, CD3, CD4, FoxP3 and PDL1(are all from BD Bioscience) various combinations cell is dyed, and use the fixed buffering of IC Liquid (eBioscience) is fixed.For dyeing in core, FoxP3 dye solution group (eBioscience) is used.In Cyan ADP 9(Beckman Coulter, Mississauga, ON) on carry out flow cytometry.

Experiment in vivo and tumor model.4T1 tumour is implanted into Balb/c mouse (Charles River Laboratories).For model in situ, by 1x10⁵A cell infusion is into second right mammary fat pad.For treating, It is put at the appointed time using insulin syringe (The Stevens Co, Montreal, QC) by (IT) or quiet in tumour (IV) injects the PBS that total volume is 100uL in arteries and veins, wherein containing 1x10⁸(plaque forming unit-pfu) a virus.Immunologic test Point inhibitor (anti-PD1(clone RMPI-14, BioXcell) and anti-CTLA 4 (cloning 9D9, BioXcell)) every other day with The dosage intraperitoneal injection (IP) of 100 μ g, total co-injection 5 times.Model is excited again for tumour, by 1 × 10⁵A cell skin bet It is mapped to the left side veutro of animal.Point processing tumour at the appointed time, and 7 days tumor resections after the first treatment.4 after operation It, by the tumour cell (3 × 10 of higher dosage⁵A cell) it is inoculated into second right fat pad.For after tumor inoculation The subgroup of the mouse excited again more than 100 days second, the interior injection 3 × 10 of bilateral fat pad⁵A EMT6 and 4T1 cell.

As a result

Pro-inflammatory signals are the needs of immune cell activated, but can generally also trigger the table of immunologic test point inhibitor (ICI) PDL1 Up to (Ritprajak, P. & Azuma, M. Intrinsic and extrinsic control of expression of the immunoregulatory molecule PD-L1 in epithelial cells and squamous cell carcinoma. Oral Oncol.51, 221–228 (2015)).In order to illustrate the mechanism of virus induction antineoplastic immune, We have carried out microarray analysis to 4T1 the and EMT6 tumour cell of external use virus or irrMG1 infection., it is surprising that Our result of study shows that irrMG1 weaker only induces a small number of genes, this forms sharp contrast with MG1, and MG1, which has been raised, to be permitted Polygenes is horizontal 300 times higher than non-infected cells.Microarray analysis also shows 4T1 the and EMT6 cell handled respectively with MG1 Raise PDL1(Figure 16 A and Figure 16 B).

In addition, removing the surface table for the culture medium induction PDL1 of virus handled through 4T1 by Flow Cytometry Assay Up to (Figure 17 A).Then, we have evaluated the regulatory T cells (CD3+, CD4+, FoxP3+ cell) in processed animal In the presence of, and observe, 10 days after virus treated, the percentage of regulatory T cells is remained unchanged in animal spleen, and in tumour The quantity of T cell then increases to from less than 40% more than 60%(Figure 17 B).In view of the success of ICI recently clinically, and it is each Kind of report shows that ICI treatment needs pre-existing anti-tumor immunity just effective, and our data showed that MG1 processing lures Lead the immune response of tumour-specificity, it is intended to determine whether the combination of two kinds of therapies can further improve result.We It attempts to combine MG1 with anti-PD1 and anti-CTLA 4 treatment.In situ in 4T1 model, it is observed that after virus treated The gross tumor volume collected substantially reduces within 12 days, and the smallest is the tumour (Figure 18 A) from the animal for receiving MG1 and ICI processing.To the greatest extent Pipe result seems to get a good chance of, but does not observe healing or survival advantage (not shown) using the therapeutic scheme.When using swollen When tumor excites model again, wherein first tumour is treated with MG1 or do not treated, second tumour is only treated with ICI, we observe 60% to the important improvement in tumour control and the group for receiving two kinds of treatments cures (Figure 18 B).This shows to use before surgery MG1 handles patient with breast cancer and generates protective immune response, can be treated in the case where recurrence by ICI and further be increased By force.

It is interesting that the increase (Figure 17 A, 17B and 18A) of PDL1 expression and regulatory T cells accumulation after MG1 processing, Provide the chance combined with ICI therapy.By reaching 60% healing (Figure 18 B) in 4T1 tumor model, it is believed that MG1- ICI combination is hopeful very much.Although not assigned it is worth noting that, ICI treatment itself reduces primary tumor burden Any survival advantage, but significantly enhance the effect of already present MG1 is induced.The discovery and various reports are consistent, show pre- The anti-tumor immune response pre-existed is necessary to effective ICI treatment.

Although cell factor and chemotactic factor (CF) are induced by virus treated, immunologic test point inhibitor (ICI) molecule PDL1 is also raised after MG1 infection by tumour cell.In view of virus treated inducing antitumor immunity response, ICI treatment is difficult to control More cancer can become sensitive now.Success is achieved in view of nearest ICI treatment, we have investigated the group with second treatment Whether close can further improve prognosis.Statistics indicate that most animals have effectively been cured in the combination of MG1 and ICI.Two kinds are controlled The combination for the treatment of improves survival rate to 60% in aggressive 4T1 TNBC mouse model.

Embodiment 4

With PDL expression in the tumor biopsy of the patient before and after oncolytic virus vaccine therapy

Background

MG1MA3 be express people MAGE-A3(transgenosis MAGE-A3 insertion) RNA oncolytic virus (Maraba Rhabdovirus MG1), there are the potentiality that cancer cell is selectively killed by least two main mechanisms.These include logical Cross the selective virus replication relative to the defective interferon response of normal cell in cancer cell.In addition to multiple in cancer cell It makes outside the virus, which is also modified to express MAGE-A3 tumor associated antigen.Therefore, host generates T to the tumour antigen Host immune system reacts to external virus protein while cellullar immunologic response.If used before delivering MG1MA3 Another virus (AdMA3；Replication defect type, the adenoviral serotype 5 of E1- and E3- missing turn with encoding human MAGE-A3 Gene) to come to the starting of MAGE-A3 tumour antigen or " causing (prime) " specific immune response, then the immune response is significant Amplification.The effect of oncolytic virus vaccine leads to MG1MA3 increase.

The test of oncolytic virus vaccine clinical

It is included in standard.With can not cure advanced stage/it is metastatic expression MAGE-A3 solid tumor patient in, start It is studied with the I/II stage with the MG1 Maraba/MAGE-A3(MG1MA3 not with adenovirus vaccine (AdMA3) together).In rank In section 1, the patient of registration has histologic study proved, unresectable Locally Advanced/metastatic entity tumor, has MAGE-A3(primary or metastatic lesion) positive expression, and without it is known extend the service life standard treatment.In the stage In II, the patient of registration has histologic study proved, unresectable Locally Advanced/metastatic entity tumor, has as follows MAGE-A3(primary or metastatic lesion) positive expression: the special sexual gland cancer of non-small cell lung cancer (NSCLC) and squamous are thin Born of the same parents' cancer, ER/PR- HER2+ breast cancer, triple negative, ER and/or PR+HER2, oesophagus/GEJ(gastroesophageal junction) cancer.

Experimental design。A group: MG1MA3(virus is administered alone).It is 1 × 10 that patient, which receives starting dose level,¹⁰Pfu's MG1MA3 was intravenously applied with the 4th day on day 1.MG1MA3 dosage gradually increases, until reaching dose limiting toxicity (DLT).B group: AdMA3(vaccine is administered alone).Patient receives in (- 14) day with 1 × 10¹⁰What the dosage of pfu was intravenously applied first exempts from AdMA3 vaccine.Do not plan to increase dosage.C group: AdMA3 adds MG1MA3(just to exempt from+reinforce).Patient (- 14) day receive with 1×10¹⁰The first of single dose intramuscular injection of pfu exempts from AdMA3 vaccine, and the MG1MA3 for then giving dosage escalation reinforces, 1st day and the 4th day 1 log initial dose with the maximal tolerance dose (MTD) lower than the recommendation determined such as in the A group of research is quiet Application in arteries and veins.G1MA3 dosage will increase, until reaching DLT in the Most patients for receiving the dosage.For A group and C group, Each dosage level at least inputs 3 patients, until reaching MTD.Core/tumor resection biopsy will before the treatment and be controlled It is carried out after treatment, and analyzes the variation of the gene expression of key signature object (including PDL1) in tumor microenvironment.

Method

According to kit protocol (Qiagen, 74704), using RNEasy Fibrous Tissue Mini Kit from core patient RNA is extracted in biopsy.In brief, using Qiagen TissueRuptor homogenizer in RLT buffer destruction group It knits.Then according to scheme, RNA is extracted using the QIAcube sample preparation object of automation.After extraction, RNA is 2100 Bioanalyzer(Agilent Technologies) on it is quantitative, then using Nanostring Elements CodeSet and NCounter 144-plex Elements TagSet analysis is up to the RNA of 100 μ g.Software is analyzed using nCounter (Nanostring Technologies) analyzes the data obtained.

As a result

Tumor biopsies inspection in two days after analyzing pretreatment by NanoString and being treated after the first time dosage of MG1 It looks into, produces clinical PDL1 expression data.NanoString analysis and observation PDL1 transcript level, is as a result expressed as treating preceding water It is flat to change with multiple horizontal after treatment, and calculate and with two divided by expression before treating by expression after treatment The different mode of kind is mapped.Figure 20 shows that A group (only Ad), B group (only MG1) and C group (Ad/MG1) are each in Present clinical test The multiple variation of PDL1 level in the individual tumors biopsy of dosage (after treatment before comparison treatment).Figure 21 is shown currently In clinical test in the combined tumor biopsy of A group (only Ad), B group (only MG1) and C group (Ad/MG1) all dosage The multiple variation of PDL1 level (after treatment before comparison treatment).Statistics indicate that MG1 and Ad/MG1 treatment causes in many patients The combination treatment using checkpoint inhibitor according to methods described herein is supported in the increase that PDL1 is expressed in tumour.

Embodiment 5

Oncolytic virus vaccine adds checkpoint inhibitor that clinical test is treated in combination

It describes in the previous patient of the Metastatic Nsclc through treating (NSCLC), and with transgenosis The MG1 Maraba/MAGE-A3(MG1MA3 of the adenovirus vaccine (AdMA3) of MAGE-A3 insertion together) (just exempting from: strengthened scheme) The clinical test of the I/II phase, multicenter, open label combined with pyridine aldoxime methyliodide (PAM) monoclonal antibody.MG1MA3 and pyridine aldoxime methyliodide (PAM) monoclonal antibody will be treated as standard Method application.

Patient will have histological subtypes squamous or non-squamous NSCLC tumour, with MAGE-A3(primary or transfer Venereal disease becomes) positive expression, patient has been completed using chemotherapeutic first standard treatment based on platinum.

Patient will receive in (- 14) day with 1 x 10¹⁰The first of dosage intramuscular (IM) application of pfu exempts from AdMA3 vaccine Single dose, and will be on day 1 with the 4th day (reinforcement) with 1 x 10¹⁰The dosage level of pfu passes through intravenous injection application MG1MA3.If the dosage combines tolerance with pyridine aldoxime methyliodide (PAM) monoclonal antibody, second group will be on day 1 with the 4th day with 1 × 10¹¹MG1MA3 into Row treatment.Patient by (- 13) day, the 8th day and later pyridine aldoxime methyliodide (PAM) monoclonal antibody is received intravenously with the dosage of 200 mg every 3 weeks, Until observing the radiology progress of confirmation.Tumor biopsy is carried out at pre-treatment and after treatment, and analyzes tumour micro-loop The variation of the gene expression of key signature object (including PDL1) in border.Objective tumor response rate (ORR) based on RECIST v1.1 It will be assessed in the 2nd stage.

Appendix A-protein and nucleotide sequence

Overall length, wild type, people MAGEA3 protein sequence (SEQ ID NO:35):

MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPD PPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLL LKYRAREPVTKAEMLGSWGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGHLYIFATCLGL SYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQ HFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHISYPPLHEWVL

REGEE*

Encoding full leng, wild type, people MAGEA3 DNA sequence dna (SEQ ID NO:36):

ATGCCTCTTGAGCAGAGGAGTCAGCACTGCAAGCCTGAAGAAGGCCTTGAGGCCCGAG GAGAGGCCCTGGGCCTGGTGGGTGCGCAGGCTCCTGCTACTGAGGAGCAGGAGGCTG CCTCCTCCTCTTCTACTCTAGTTGAAGTCACCCTGGGGGAGGTGCCTGCTGCCGAGTCA CCAGATCCTCCCCAGAGTCCTCAGGGAGCCTCCAGCCTCCCCACTACCATGAACTACC CTCTCTGGAGCCAATCCTATGAGGACTCCAGCAACCAAGAAGAGGAGGGGCCAAGCAC CTTCCCTGACCTGGAGTCCGAGTTCCAAGCAGCACTCAGTAGGAAGGTGGCCGAGTTG GTTCATTTTCTGCTCCTCAAGTATCGAGCCAGGGAGCCGGTCACAAAGGCAGAAATGCT GGGGAGTGTCGTCGGAAATTGGCAGTATTTCTTTCCTGTGATCTTCAGCAAAGCTTCCA GTTCCTTGCAGCTGGTCTTTGGCATCGAGCTGATGGAAGTGGACCCCATCGGCCACTT GTACATCTTTGCCACCTGCCTGGGCCTCTCCTACGATGGCCTGCTGGGTGACAATCAGA TCATGCCCAAGGCAGGCCTCCTGATAATCGTCCTGGCCATAATCGCAAGAGAGGGCGA CTGTGCCCCTGAGGAGAAAATCTGGGAGGAGCTGAGTGTGTTAGAGGTGTTTGAGGGG AGGGAAGACAGTATCTTGGGGGATCCCAAGAAGCTGCTCACCCAACATTTCGTGCAGG AAAACTACCTGGAGTACCGGCAGGTCCCCGGCAGTGATCCTGCATGTTATGAATTCCTG TGGGGTCCAAGGGCCCTCGTTGAAACCAGCTATGTGAAAGTCCTGCACCATATGGTAAA GATCAGTGGAGGACCTCACATTTCCTACCCACCCCTGCATGAGTGGGTTTTGAGAGAG GGGGAAGAGTGA

Encoding full leng, wild type, people's MAGEA3 albumen codon optimization DNA sequence dna (SEQ ID NO:37):

ATGCCCCTGGAGCAGCGGTCTCAGCATTGCAAGCCAGAGGAGGGCCTCGAGGCGAGG GGCGAGGCCCTCGGCTTGGTGGGGGCGCAGGCTCCTGCAACCGAGGAGCAAGAGGC CGCATCCAGTTCCTCTACCCTGGTTGAGGTGACCTTGGGTGAGGTGCCCGCCGCGGAG AGCCCCGACCCGCCTCAAAGCCCCCAGGGTGCCAGCTCCCTGCCCACAACAATGAACT ACCCACTCTGGAGTCAGTCTTACGAGGACAGTAGTAACCAAGAGGAGGAGGGACCCTC CACATTCCCAGACCTGGAGTCTGAATTCCAGGCAGCATTGTCTAGAAAAGTGGCCGAAT TGGTGCACTTCCTGCTGCTGAAGTATCGCGCCCGCGAGCCAGTCACAAAAGCTGAAAT GCTGGGTTCTGTCGTGGGAAATTGGCAGTACTTCTTCCCCGTGATCTTCAGTAAAGCGT CCAGCTCCTTGCAGCTGGTCTTTGGTATCGAGCTGATGGAGGTGGATCCCATCGGCCA TCTGTATATCTTTGCCACATGCCTGGGCCTGAGCTACGATGGCCTGCTGGGCGACAAC CAGATCATGCCAAAAGCTGGCCTGCTGATCATCGTTCTGGCTATCATCGCTAGAGAAGG AGATTGCGCCCCTGAAGAAAAGATCTGGGAGGAACTGAGCGTCCTGGAAGTCTTTGAG GGTCGTGAAGACAGCATTCTCGGGGATCCCAAGAAGCTGCTGACCCAGCACTTCGTGC AGGAGAACTATCTGGAGTACCGCCAGGTTCCCGGCAGCGACCCCGCTTGCTACGAGTT CCTGTGGGGCCCCAGGGCCCTGGTCGAGACATCCTACGTGAAGGTCCTGCACCATATG GTTAAAATCAGCGGCGGCCCCCATATCTCTTATCCGCCGCTCCACGAGTGGGTGCTCC GGGAGGGAGAGGAG

Overall length, wild type, people MAGEA3 variant protein sequence (SEQ ID NO:38):

MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPD PPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLL LKYRAREPVTKAEMLGSWGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGHLYIFATCLGL SYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQ HFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHISYPPLHEWVL REGEEDYKDDDDK*

Encoding full leng, wild type, people MAGEA3 variant DNA sequence dna (SEQ ID NO:39):

ATGCCCCTGGAACAGCGGAGCCAGCACTGCAAGCCCGAGGAAGGCCTGGAAGCCAGA GGCGAAGCCCTGGGACTGGTGGGAGCCCAGGCCCCTGCCACAGAAGAACAGGAAGCC GCCAGCAGCAGCTCCACCCTGGTGGAAGTGACCCTGGGCGAAGTGCCTGCCGCCGAG AGCCCTGATCCCCCTCAGTCTCCTCAGGGCGCCAGCAGCCTGCCCACCACCATGAACT ACCCCCTGTGGTCCCAGAGCTACGAGGACAGCAGCAACCAGGAAGAGGAAGGCCCCA GCACCTTCCCCGACCTGGAAAGCGAGTTCCAGGCCGCCCTGAGCCGGAAGGTGGCAG AGCTGGTGCACTTCCTGCTGCTGAAGTACAGAGCCCGCGAGCCCGTGACCAAGGCCGA GATGCTGGGCAGCGTGGTGGGAAACTGGCAGTACTTCTTCCCCGTGATCTTCTCCAAG GCCAGCAGCTCCCTGCAGCTGGTGTTCGGCATCGAGCTGATGGAAGTGGACCCCATCG GCCACCTGTACATCTTCGCCACCTGTCTGGGCCTGAGCTACGACGGCCTGCTGGGCGA CAACCAGATCATGCCCAAGGCCGGCCTGCTGATCATCGTGCTGGCCATCATTGCCCGC GAGGGCGACTGCGCCCCTGAGGAAAAGATCTGGGAGGAACTGAGCGTGCTGGAAGTG TTCGAGGGCAGAGAGGACAGCATCCTGGGCGACCCCAAGAAGCTGCTGACCCAGCAC TTCGTGCAGGAAAACTACCTGGAATACCGCCAGGTGCCCGGCAGCGACCCCGCCTGTT ACGAGTTCCTGTGGGGCCCCAGGGCTCTGGTGGAAACCAGCTACGTGAAGGTGCTGCA CCACATGGTGAAAATCAGCGGCGGACCCCACATCAGCTACCCCCCACTGCACGAGTGG GTGCTGAGAGAGGGCGAAGAGGACTACAAGGACGACGACGACAAATGA

The protein sequence (SEQ ID NO:40) of HPV E6/E7 fusion protein:

MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYR DGNPYAVDKLKFYSKISEYRHYCYSVYGTTLEQQYNKPLCDLLIRINQKPLCPEEKQRHLDK KQRFHNIRGRWTGRCMSCCRSSRTRRETQLGGGGGAAYMARFEDPTRRPYKLPDLCTEL NTSLQDIEITCVYCKTVLELTEVFEFAFKDLFWYRDSIPHAAHKIDFYSRIRELRHYSDSVYG DTLEKLTNTGLYNLLIRLRQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERL QRRRETQVGGGGGAAYMHGDTPTLHEYMLDLQPETTDLYQLNDSSEEEDEIDGPAGQAEP DRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVPICSQKPGGGGGAAYMHGP KATLQDIVLHLEPQNEIPVDLLQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCE ARIKLWESSADDLRAFQQLFLNTLSFVPWCASQQ*

The DNA sequence dna (SEQ ID NO:41) of HPV E6/E7 fusion protein:

ATGCATCAGAAGCGAACTGCTATGTTTCAGGACCCTCAGGAGCGGCCACGCAAACTGC CTCAGCTGTGCACCGAACTGCAGACAACTATCCACGACATCATTCTGGAATGCGTGTAC TGTAAGCAGCAGCTGCTGAGGAGAGAGGTCTATGACTTCGCTTTTCGCGATCTGTGCAT CGTGTACCGAGACGGAAACCCATATGCAGTCGATAAGCTGAAGTTCTACAGCAAGATCT CCGAATACAGGCATTACTGTTACAGCGTGTACGGGACCACACTGGAGCAGCAGTATAAC AAGCCCCTGTGCGACCTGCTGATCAGAATTAATCAGAAGCCCCTGTGCCCTGAGGAAAA ACAGAGGCACCTGGATAAGAAACAGAGATTTCATAACATCCGAGGACGATGGACCGGG CGGTGCATGTCCTGCTGTAGAAGCTCCCGGACTCGACGAGAGACCCAGCTGGGCGGA GGAGGAGGAGCAGCTTACATGGCACGATTCGAGGACCCTACCCGAAGGCCATATAAGC TGCCCGACCTGTGCACAGAACTGAATACTTCTCTGCAGGACATCGAGATTACATGCGTG TACTGTAAAACCGTCCTGGAGCTGACAGAAGTGTTCGAGTTTGCTTTCAAGGACCTGTT TGTGGTCTACCGGGATTCAATCCCTCACGCAGCCCATAAAATCGACTTCTACAGCAGGA TCAGGGAACTGCGCCACTACTCCGACAGCGTGTACGGGGATACACTGGAGAAGCTGAC AAACACTGGCCTGTACAATCTGCTGATCCGACTGCGACAGAAGCCACTGAACCCAGCC GAAAAACTGAGACACCTGAACGAGAAGAGACGGTTTCACAATATTGCAGGCCATTATAG GGGACAGTGCCATAGTTGCTGTAATCGAGCCAGGCAGGAAAGACTGCAGCGCCGAAG GGAGACTCAAGTCGGCGGAGGAGGAGGAGCTGCATACATGCACGGCGACACCCCCAC ACTGCATGAATATATGCTGGATCTGCAGCCTGAGACTACCGACCTGTACCAGCTGAACG ATTCTAGTGAGGAAGAGGACGAAATCGACGGACCAGCAGGACAGGCAGAGCCTGACC GGGCCCACTATAATATTGTGACATTCTGCTGTAAGTGCGATTCTACTCTGCGGCTGTGC GTGCAGAGTACTCATGTCGACATCCGCACCCTGGAGGATCTGCTGATGGGGACTCTGG GCATCGTCCCAATTTGTAGCCAGAAACCAGGCGGCGGCGGCGGAGCAGCTTACATGCA CGGACCCAAGGCTACCCTGCAGGACATCGTGCTGCATCTGGAACCTCAGAATGAGATT CCAGTCGACCTGCTGCAGCTGAGTGATTCAGAAGAGGAAAACGACGAGATCGACGGCG TGAATCACCAGCATCTGCCTGCTAGACGGGCAGAGCCACAGCGACACACAATGCTGTG CATGTGCTGTAAGTGTGAAGCCAGGATCAAGCTGGTGGTCGAGTCAAGCGCCGACGAT CTGCGCGCCTTCCAGCAGCTGTTCCTGAATACTCTGTCATTTGTCCCTTGGTGTGCCTC CCAGCAGTGA

The protein sequence (SEQ ID NO:42) of huSTEAP albumen:

MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSEL QHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLAL VYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRY KLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYI QSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIWLIFKSILFLPCLRKKILKIR HGWEDVTKINKTEICSQLKL*

The DNA sequence dna (SEQ ID NO:43) of huSTEAP albumen:

ATGGAATCACGGAAGGACATCACTAATCAGGAGGAACTGTGGAAAATGAAGCCAAGAA GGAATCTGGAAGAGGACGACTATCTGCACAAGGACACCGGCGAAACAAGTATGCTGAA ACGACCAGTGCTGCTGCACCTGCATCAGACTGCTCACGCAGACGAGTTTGATTGCCCC TCTGAACTGCAGCACACCCAGGAGCTGTTCCCACAGTGGCATCTGCCCATCAAGATTGC CGCTATCATTGCTTCACTGACATTTCTGTACACTCTGCTGAGAGAAGTGATCCACCCCCT GGCCACCAGCCATCAGCAGTACTTCTATAAGATCCCTATCCTGGTCATCAACAAGGTCC TGCCAATGGTGAGCATCACACTGCTGGCCCTGGTCTACCTGCCTGGAGTGATCGCAGC CATTGTCCAGCTGCACAATGGGACAAAGTATAAGAAATTTCCACATTGGCTGGATAAGT GGATGCTGACTAGGAAACAGTTCGGACTGCTGTCCTTCTTTTTCGCCGTGCTGCACGCT ATCTACAGCCTGTCCTATCCCATGAGGAGGAGCTACCGGTATAAGCTGCTGAACTGGG CTTACCAGCAGGTGCAGCAGAACAAGGAGGACGCATGGATTGAACATGACGTGTGGCG CATGGAAATCTACGTGAGCCTGGGCATTGTCGGACTGGCCATCCTGGCTCTGCTGGCA GTGACCAGTATCCCTTCTGTCAGTGACTCACTGACATGGAGAGAGTTTCACTACATTCA GAGCAAGCTGGGGATCGTGTCCCTGCTGCTGGGCACCATCCATGCACTGATTTTTGCC TGGAACAAGTGGATCGATATCAAGCAGTTCGTGTGGTATACTCCCCCTACCTTTATGATT GCCGTCTTCCTGCCCATCGTGGTCCTGATCTTCAAGTCCATCCTGTTCCTGCCTTGTCT GCGGAAGAAAATCCTGAAAATTCGGCACGGATGGGAGGATGTCACCAAAATCAATAAGA CTGAAATCTGTAGCCAGCTGAAGCTTTAA

The protein sequence (SEQ ID NO:44) of NYESQ1 MAR albumen:

MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPG

GGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLP

VPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR*

The DNA sequence dna (SEQ ID NO:45) of NYES01 MAR:

ATGCAGGCCGAGGGCAGAGGCACAGGCGGATCTACAGGCGACGCCGATGGCCCTGGC GGCCCTGGAATTCCTGACGGACCTGGCGGCAATGCCGGCGGACCCGGAGAAGCTGGC GCCACAGGCGGAAGAGGACCTAGAGGCGCTGGCGCCGCTAGAGCTTCTGGACCAGGC GGAGGCGCCCCTAGAGGACCTCATGGCGGAGCCGCCTCCGGCCTGAACGGCTGTTGC AGATGTGGAGCCAGAGGCCCCGAGAGCCGGCTGCTGGAATTCTACCTGGCCATGCCCT TCGCCACCCCCATGGAAGCCGAGCTGGCCAGACGGTCCCTGGCCCAGGATGCTCCTC CTCTGCCTGTGCCCGGCGTGCTGCTGAAAGAATTCACCGTGTCCGGCAACATCCTGAC CATCCGGCTGACTGCCGCCGACCACAGACAGCTCCAGCTGTCTATCAGCTCCTGCCTG CAGCAGCTGAGCCTGCTGATGTGGATCACCCAGTGCTTTCTGCCCGTGTTCCTGGCTC AGCCCCCCAGCGGCCAGAGAAGATGA

Claims

1. a kind for the treatment of and/or pre- anti-cancer in mammal in need or the method for extending antitumor reaction, the side Method includes that a effective amount of combination is applied to the mammal, oncolytic rhabdovirus and (b) of the combination comprising (a) replicability One or more checkpoint inhibitor.

2. according to the method described in claim 1, wherein the checkpoint inhibitor be monoclonal antibody, it is humanized antibody, complete Human antibody, fusion protein or combinations thereof.

3. according to the method described in claim 1, wherein the checkpoint inhibitor inhibits checkpoint albumen selected from the following: thin Cellular toxicity T- lymphocyte antigen -4(CTLA4), apoptosis albumen 1(PD-1), PD-L1, PD-L2, B7-H3, B7- H4, herpesviral enter regulator (HVEM), T cell memebrane protein 3(TIM3), galactose agglutinin 9(GAL9), lymphocyte it is living Change gene 3(LAG3), containing V structure domain immunoglobulin (Ig) T cell activation repressor (VISTA), killing cell exempt from Epidemic disease globulin sample receptor (KIR), B and T lymphocyte attenuator (BTLA), T cell with Ig and ITIM structural domain it is immune by Body (TIGIT), and combinations thereof.

4. according to the method described in claim 3, wherein the checkpoint inhibitor inhibits CTLA-4, PD-1 or PD-L1.

5. according to the method described in claim 4, wherein the checkpoint inhibitor inhibits CTLA-4 and is selected from Yi Pulimu Ma (Ipilimumab) and Tremelimumab.

6. according to the method described in claim 4, wherein the checkpoint inhibitor inhibits PD-1 and selected from receiving military monoclonal antibody (Nivolumab), pyridine aldoxime methyliodide (PAM) monoclonal antibody (Pembrolizumab), Pidilizumab, lambrolizumab and AMP-224.

7. according to the method described in claim 4, wherein the checkpoint inhibitor inhibits PD-L1 and is selected from BMS- 936559, MEDI-4736, MPDL33280A, M1H1, Aunar Zhu monoclonal antibody (Atezolizumab), Durvalumab and Avelumab。

8. -7 described in any item methods according to claim 1, wherein the oncolytic rhabdovirus and at least two checkpoints Inhibitor is administered in combination to the mammal.

9. -8 described in any item methods according to claim 1, wherein the oncolytic rhabdovirus and the checkpoint inhibit Agent is administered simultaneously.

10. -8 described in any item methods according to claim 1, wherein the oncolytic rhabdovirus and the checkpoint inhibit Agent is sequentially applied, and the first time application that wherein first time of checkpoint inhibitor is applied in oncolytic virus occurs before, And it is preferred that occurring within 30 days of the first time application of oncolytic virus.

11. according to described in any item methods of preceding claims, wherein oncolytic rhabdovirus expression tumour correlation is anti- It is former.

12. according to the method for claim 11, wherein the tumor associated antigen is selected from MAGEA3, human papilloma virus Six cross-film epithelium antigens, Cancer Testis Antigens 1 and its variant of E6/E7 fusion protein, human prostate albumen.

13. method according to claim 11 or 12, wherein the mammal has to the tumor associated antigen Pre-existing immunity.

14. according to the method for claim 13, wherein immunity pre-existing in the mammal is by applying The tumor associated antigen is applied with the forward direction mammal of oncolytic rhabdovirus to establish.

15. according to the method for claim 14, wherein immunity pre-existing in the mammal is by applying The expression vector of the tumor associated antigen is encoded with the application of the forward direction mammal of oncolytic rhabdovirus to establish.

16. according to the method for claim 15, wherein the expression vector is selected from adenovirus vector, poxvirus vector, inverse Transcription vector, α viral vectors, plasmid and load antigen presenting cell.

17. according to described in any item methods of preceding claims, wherein the oncolytic rhabdovirus is oncolytic hydroa Poison.

18. according to the method for claim 17, the oncolytic rhabdovirus be wild type or VSV through gene modification or Maraba bacterial strain rhabdovirus.

19. according to the method for claim 17, wherein the oncolytic rhabdovirus is VSVdelta51 or Maraba MG1.

20. according to the method for claim 14, wherein the oncolytic rhabdovirus is Maraba MG1.

21. according to described in any item methods of preceding claims, wherein the oncolytic rhabdovirus is as 10⁶-10¹⁴pfu、 10⁶-10¹²pfu、10⁸-10¹⁴pfu、10⁸-10¹²Or 10¹⁰-10¹²One or more dosage application in pfu.

22. according to described in any item methods of preceding claims, wherein the oncolytic rhabdovirus is applied intravascularly.

23. according to described in any item methods of preceding claims, wherein the cancer is colorectal cancer, lung cancer, melanocyte Tumor, cancer of pancreas, oophoroma, clear-cell carcinoma, cervical carcinoma, liver cancer, breast cancer, head and neck cancer, prostate cancer, gastroesophageal junction cancer, The cancer of the brain and soft tissue sarcoma.

24. according to the method for claim 23, wherein the cancer is ER/PR- HER2+ breast cancer, three negative breasts Cancer, ER and/or PR+ HER2+ breast cancer, squamous or non-squamous non-small cell lung cancer (NSCLC) or gastroesophageal junction cancer.

25. according to described in any item methods of preceding claims, wherein the checkpoint inhibitor is antibody or fusion egg It is white, and as 0.01-10 mg/kg, 0.1-10 mg/kg, 1-10 mg/kg, 2-8 mg/kg, 3-7 mg/kg, 4-5 mg/ One or more dosage application in kg or at least 10 mg/kg.

26. according to the method for claim 24, wherein the checkpoint inhibitor is administered weekly at least three times, weekly extremely Few four times, at least five times weekly, once a week, once every two weeks, it is primary every other week or once every three weeks.

27. according to described in any item methods of preceding claims, wherein the mammal is people.

28. described in any item methods of 1-22 and 24 according to claim 1, the cancer expresses tumor associated antigen.

29. according to the method for claim 27, wherein the tumor associated antigen is MAGE-A3.

30. a kind for the treatment of and/or pre- anti-cancer in the mankind in need or the method for extending antitumor reaction, the method packet Include to expression Cancer Testis Antigens melanoma antigen family A3(MAGE-A3) a effective amount of group of human administration of cancer It closes, Maraba MG1 and (b) PD-1 inhibitor of the combination comprising (a) expression MAGE-A3.

31. the method according to claim 27, wherein the cancer is ER/PR- HER2+ breast cancer, three negative breasts Cancer, ER and/or PR+ HER2+ breast cancer, squamous or non-squamous NSCLC or gastroesophageal junction cancer.

32. the method according to claim 29 or 30, wherein the PD-1 inhibitor is pyridine aldoxime methyliodide (PAM) monoclonal antibody (pembrolizumab).

33. according to described in any item methods of claim 29-31, wherein expressing the excellent of the Maraba MG1 of MAGE-A3 Before selecting intravenous first time to apply, the adenovirus vector of expression MAGE-A3 is preferably intramuscularly applied to the mankind Single just exempts from dosage about 1 to 3 week, preferably from about 2 weeks.

34. according to described in any item methods of claim 29-32, wherein with 10¹⁰To 10¹²Pfu, preferably 10¹⁰Or 10¹¹ The dosage application Maraba MG1 of pfu is one or many.

35. the method according to claim 32 or 33, wherein exempting from agent at the beginning of expressing the single of adenovirus vector of MAGE-A3 After amount, and before the first time dosage of the Maraba MG1 in expression MAGE-A3, the first time agent of PD-1 inhibitor is applied Amount.

36. according to described in any item methods of claim 29-34, wherein in the chemotherapy by preferably including platinum duplex therapy At least one period treated after, the cancer has had progressed.